A two-photon fluorescent probe based on 2,1,3-benzothiadiazole that rapidly responds to cysteine, as well as its preparation method and application

Abstract

The present invention discloses a two-photon fluorescent probe based on 2,1,3-benzothiadiazole that quickly responds to cysteine, as well as a preparation method and application thereof. The present invention synthesizes the two-photon fluorescent probe F-BTD, a fast-response cysteine ​​probe based on 2,1,3-benzothiadiazole, in three steps. The two-photon fluorescent probe F-BTD has two-photon performance and can be used for rapid and sensitive quantitative detection of cysteine. It can also perform fluorescence imaging of exogenous and endogenous cysteine ​​in HeLa cells, and has the capability of two-photon fluorescence imaging of exogenous and endogenous cysteine ​​in cells.

Description

Technical Field

[0001] The present invention belongs to the field of small molecule fluorescent probes, and specifically relates to a two-photon fluorescent probe based on 2,1,3-benzothiadiazole that quickly responds to cysteine, and a preparation method and application thereof. Background Art

[0002] Cysteine ​​is an important endogenous thiol in organisms and is considered a key biomolecule for maintaining redox homeostasis. The intracellular concentration of cysteine ​​varies between 30 and 200 μM, and the total plasma concentration is approximately 250 μM. Under normal circumstances, the production, consumption, accumulation, and elimination of cysteine ​​in living systems are in a dynamic equilibrium. Fluctuations in its concentration are associated with a variety of diseases, including neurodegenerative diseases such as Alzheimer's and Parkinson's disease, cardiovascular disease, liver damage, and epilepsy. Therefore, it is necessary to monitor and quantify the concentration levels of cysteine ​​in biological systems to assess its distribution and metabolic characteristics, which is crucial for understanding the role of cysteine ​​in chemistry and biology.

[0003] In recent years, methods such as high-performance liquid chromatography, mass spectrometry, and capillary zone electrophoresis have been widely used for cysteine ​​determination, with fluorescence-based detection of cysteine ​​experiencing rapid development. Importantly, cysteine-responsive fluorescent probes offer powerful analytical and discriminatory capabilities, improving detection sensitivity and speed. Furthermore, fluorescent probes, due to their high spatiotemporal resolution and sensitivity, have become recognized as powerful tools for cysteine ​​measurement, particularly in in vivo analysis. To date, most cysteine-responsive fluorescent probes are based on the redox and nucleophilic properties of cysteine. The fluorescence sensing mechanisms employed primarily include acrylate cyclization, aldehyde cyclization, and Michael addition reactions. Currently reported fluorescent precursors for cysteine ​​detection primarily focus on coumarins, quinolines, naphthalimides, fluorescein, and BODIPY. Therefore, continuous efforts are needed to utilize novel fluorescent precursors for cysteine ​​detection to improve imaging depth and signal-to-noise ratio in biological systems. Summary of the Invention

[0004] The present invention aims to provide a two-photon fluorescent probe based on 2,1,3-benzothiadiazole that quickly responds to cysteine ​​and a preparation method thereof. The two-photon fluorescent probe has fast reaction speed, strong specificity, good sensitivity and excellent two-photon performance.

[0005] Another object of the present invention is to provide the application of the 2,1,3-benzothiadiazole-based two-photon fluorescent probe that quickly responds to cysteine ​​in the quantitative detection of cysteine. When used for quantitative detection of cysteine ​​concentration, it has high sensitivity and a detection limit as low as 232nM.

[0006] Another object of the present invention is to provide the application of the 2,1,3-benzothiadiazole-based two-photon fluorescent probe that quickly responds to cysteine ​​in cell fluorescence imaging for non-disease diagnosis and treatment purposes, which can realize fluorescence imaging of endogenous and exogenous cysteine ​​in HeLa cells, as well as two-photon fluorescence imaging of exogenous cysteine ​​in HeLa cells.

[0007] To achieve the above-mentioned purpose, the technical solutions adopted by the present invention are as follows:

[0008] The present invention provides a two-photon fluorescent probe based on 2,1,3-benzothiadiazole that quickly responds to cysteine, characterized in that the structural formula of the two-photon fluorescent probe is:

[0009]

[0010] The present invention also provides a method for preparing the 2,1,3-benzothiadiazole-based fast-response cysteine ​​two-photon fluorescent probe, comprising the following steps:

[0011] (1) 4,7-dibromo-2,1,3-benzothiadiazole, 4-hydroxyphenylboronic acid pinacol ester, tetrabutylammonium bromide, and tetrakis(triphenylphosphine)palladium are added to a mixed solution of toluene and ethanol, and then a potassium carbonate solution is added. Under the protection of an inert gas, the mixture is stirred at 75 to 85° C. for 20 to 24 hours, and then the reaction solution is extracted, concentrated, purified, and dried to obtain a monohydroxy-substituted 2,1,3-benzothiadiazole compound BTD-Br. The structural formula of the BTD-Br is:

[0012] (2) Substituting BTD-Br for 4,7-dibromo-2,1,3-benzothiadiazole in step (1), repeating step (1) to synthesize a dihydroxy-substituted 2,1,3-benzothiadiazole fluorescent matrix BTD-OH, wherein the structural formula of BTD-OH is:

[0013] (3) BTD-OH and 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) are dissolved in anhydrous acetonitrile, and then potassium carbonate is added and stirred at room temperature for 6 to 8 hours. After the reaction is completed, the mixture is centrifuged, washed, and dried to obtain the 2,1,3-benzothiadiazole-based two-photon fluorescent probe F-BTD that rapidly responds to cysteine. In step (1), the molar ratio of 4,7-dibromo-2,1,3-benzothiadiazole, 4-hydroxyphenylboronic acid pinacol, tetrabutylammonium bromide, and tetrakis(triphenylphosphine)palladium is 1:1.2 to 1.5:0.05 to 0.06:0.01 to 0.02.

[0014] In step (1), the volume ratio of toluene to ethanol is 3:2; the concentration of 4,7-dibromo-2,1,3-benzothiadiazole in the mixed solution of toluene and ethanol is 0.1-0.2 mol / L.

[0015] In step (1), the concentration of the potassium carbonate solution is 1.5 to 2.5 mol / L; and the volume ratio of the potassium carbonate solution to the mixed solution of toluene and ethanol is 1:5.

[0016] In step (1), the purification method is: using ethyl acetate: petroleum ether = 1:3-5 as eluent, purifying the product through a silica gel column, collecting the product and concentrating it.

[0017] In step (2), the molar ratio of BTD-Br, 4-hydroxyphenylboronic acid pinacol, tetrabutylammonium bromide, and tetrakis(triphenylphosphine)palladium is 1:1.2-1.5:0.05-0.06:0.01-0.02.

[0018] In step (2), the crude product is purified by using ethyl acetate: petroleum ether = 1:2-4 as eluent, collecting the product through a silica gel column and concentrating it.

[0019] In step (3), the molar ratio of BTD-OH, NBD-Cl and potassium carbonate is 1:2.2-2.5:3-5.

[0020] In step (3), the concentration of NBD-Cl in the acetonitrile solution is 0.01 to 0.02 mol / L.

[0021] The present invention also provides the use of the 2,1,3-benzothiadiazole-based two-photon fluorescent probe that quickly responds to cysteine ​​in the quantitative detection of cysteine.

[0022] The present invention also provides the use of the two-photon fluorescent probe based on 2,1,3-benzothiadiazole rapid response cysteine ​​in cell fluorescence imaging for non-disease diagnosis and treatment purposes.

[0023] The present invention adopts a three-step synthesis method to simply and conveniently synthesize a fluorescent probe F-BTD based on 2,1,3-benzothiadiazole rapid response cysteine. In its structure, dihydroxy substituted 2,1,3-benzothiadiazole is a fluorophore, nitro-2,1,3-benzoxadiazole (NBD-Cl) is a recognition group, and the two are connected by an ether bond. The recognition group NBD in the structure has a strong electron-withdrawing ability, so the probe molecule F-BTD fluorescence is very weak. However, when cysteine ​​is added, the ether bond breaks due to the strong reducing ability of cysteine, releasing the fluorescent matrix and enhancing the fluorescence of the system.

[0024] In vitro experimental results demonstrated that F-BTD responds to cysteine ​​within 2 minutes and exhibits two-photon performance, with a two-photon absorption cross section of 93 GM. Furthermore, F-BTD exhibits excellent specificity, showing no significant response to metal ions, amino acids, other biothiols, or reducing substances in biological systems. Cell imaging experiments demonstrated that F-BTD can perform fluorescence imaging of endogenous and exogenous cysteine ​​in HeLa cells. More importantly, it can also perform two-photon fluorescence imaging of exogenous cysteine ​​in HeLa cells. Therefore, F-BTD holds great promise for application in cysteine-related research and monitoring.

[0025] Compared with the prior art, the 2,1,3-benzothiadiazole-based fast-response cysteine ​​two-photon fluorescent probe F-BTD provided by the present invention has a fast reaction speed, strong specificity, good sensitivity and excellent two-photon performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the structural formula of probe F-BTD;

[0027] Figure 2 This is the synthetic route of the probe F-BTD;

[0028] Figure 3 is the H NMR spectrum of BTD-Br;

[0029] Figure 4 is the H NMR spectrum of BTD-OH;

[0030] Figure 5 is the H NMR spectrum of probe F-BTD;

[0031] Figure 6 The UV absorption spectrum (A) and fluorescence emission spectrum (B) of the probe F-BTD and the reaction of the probe F-BTD and cysteine ​​(Cys) in Example 2 are shown;

[0032] Figure 7 : is the curve of the change of the fluorescence emission intensity of the probe F-BTD and the probe F-BTD after the reaction with cysteine ​​(Cys) in Example 2 as a function of pH, and the pH value range is 2 to 12;

[0033] Figure 8 Graph showing the relationship between the fluorescence emission intensity of the probe F-BTD and cysteine ​​(Cys) after reaction with time in Example 2;

[0034] Figure 9 Figure 2 shows the fluorescence emission spectra of the probe F-BTD after reacting with different concentrations of cysteine ​​(Cys) (A) and the linear relationship between the fluorescence emission intensity and the cysteine ​​(Cys) concentration (B);

[0035] Figure 10 is the two-photon performance diagram of the probe F-BTD;

[0036] Figure 11 The fluorescence intensity of the probe F-BTD specifically recognizes cysteine ​​(Cys) and the multiple relationship between the difference between the blank control group and the blank group. The analytes in the figure are 1. control group, 2. Val, 3. Trp, 4. Arg, 5. His, 6. Asp, 7. Glu, 8. Na + , 9.K + 、10.Ca 2+ 、11.Mg 2+ 、12.Zn 2+ 、13.Cu 2+ 、14.Fe 2+ 、15.Fe 3+ , 16.NaClO, 17.NaNO2, 18.H2O2, 19.NaHSO3, 20.Na2S, 21.AA, 22.Cys;

[0037] Figure 12 is the cytotoxicity of the probe F-BTD;

[0038] Figure 13 Diagram of the mechanism of probe F-BTD responding to cysteine ​​(Cys);

[0039] Figure 14 is the mass spectrum of probe F-BTD;

[0040] Figure 15 is the mass spectrum of the reaction between probe F-BTD and cysteine ​​(Cys);

[0041] Figure 16 This is the fluorescence imaging of probe F-BTD on exogenous cysteine ​​(Cys) in HeLa cells;

[0042] Figure 17 This is the fluorescence imaging of the probe F-BTD on endogenous cysteine ​​(Cys) in HeLa cells; Figure 18 This is the two-photon fluorescence imaging of probe F-BTD on exogenous cysteine ​​(Cys) in HeLa cells. DETAILED DESCRIPTION

[0043] Example 1

[0044] A two-photon fluorescent probe based on 2,1,3-benzothiadiazole that rapidly responds to cysteine, the structural formula of which is:

[0045]

[0046] The synthetic route of the cysteine-responsive fluorescent probe based on 2,1,3-benzothiadiazole is as follows: Figure 2 As shown, its synthesis method comprises the following steps:

[0047] (1) Synthesis of compound BTD-Br: Weigh 0.5830 g (2 mmol) of 4,7-dibromo-2,1,3-benzothiadiazole and 0.572 g (2.6 mmol) of 4-hydroxyphenylboronic acid pinacol ester, then weigh 0.0310 g (0.1 mmol) of tetrabutylammonium bromide and 0.0392 g (0.03 mmol) of tetrakis(triphenylphosphine)palladium, add them to 9 mL of toluene and 6 mL of anhydrous ethanol, and finally add 3 mL of 2 mol / L (0.6415 g) of potassium carbonate solution. Mix well, evacuate the air from the round-bottom flask, and reflux at 75°C for 24 hours under nitrogen atmosphere. After the reaction, cool naturally to room temperature and quickly pour into 100 mL of water to quench the reaction. Then, extract three times with 50 mL of dichloromethane, wash three times with 50 mL of water, and then wash three times with 50 mL of saturated brine. After drying over anhydrous magnesium sulfate, the mixture was rotary evaporated to remove dichloromethane and purified on a silica gel column using ethyl acetate: petroleum ether = 1:4 (V:V) as eluent. After vacuum drying, the intermediate BTD-Br was obtained, and its H NMR spectrum was as follows: Figure 3 As shown, 1 H NMR (400MHz, DMSO-d6) δ9.82 (s, 1H), 8.08 (d, J = 7.7Hz, 1H), 7.84 (d, J = 8.6Hz, 2H), 7.69 (d, J = 7.6Hz, 1H), 6.93 (d, J = 8.6Hz, 2H).

[0048] (2) Synthesis of compound BTD-OH: The synthesis steps and pretreatment methods are the same as those in step 1, except that the 4,7-dibromo-2,1,3-benzothiadiazole in step (1) is replaced by an equal amount of BTD-Br; the purification method is: purification on a silica gel column with an eluent of ethyl acetate: petroleum ether = 1:2 (V / V). After vacuum drying, the fluorescent matrix BTD-OH is obtained, and its H NMR spectrum is as follows: Figure 4 As shown, 1 H NMR (400MHz, DMSO-d6) δ9.75 (s, 2H), 7.92–7.84 (m, 4H), 7.81 (s, 2H), 6.93 (d, J = 8.7Hz, 4H).

[0049] (3) Synthesis of fluorescent probe F-BTD: Weigh 0.0141 g (0.044 mmol) of BTD-OH and 0.0183 g (0.098 mmol) of 4-chloro-7-nitro-2,1,3-benzothiadiazole (NBD-Cl) and add them to 7 mL of anhydrous acetonitrile, and add 0.0195 g (0.141 mmol) of potassium carbonate as catalyst, and stir at room temperature for 6 hours. After the reaction is completed, centrifuge at 8000 rpm for 10 minutes to obtain a precipitate. Wash the precipitate three times with water and freeze-dry to obtain a two-photon fluorescent probe F-BTD based on 2,1,3-benzothiadiazole that responds quickly to cysteine. Its nuclear magnetic hydrogen spectrum is as follows Figure 5 shown. 1 H NMR (400MHz, DMSO-d6) δ8.71 (d, J = 8.4Hz, 2H), 8.33-8.25 (m, 4H), 8.11 (s, 2H), 7.65 (d, J = 8.7Hz, 4H), 6.89 (d, J = 8.4Hz, 2H).

[0050] Example 2

[0051] Response performance of probe F-BTD to cysteine

[0052] 20 μL of 500 μM probe F-BTD dimethyl sulfoxide solution was added to 980 μL of 20 mM phosphate buffer solution with a pH of 7.40 to obtain solution A;

[0053] 20 μL of a 500 μM dimethyl sulfoxide solution of the probe F-BTD was added to 930 μL of a 20 mM phosphate buffer solution with a pH of 7.40, followed by the addition of 50 μL of a 10 mM cysteine ​​solution to obtain solution B. The final concentration of cysteine ​​in solution B was 500 μM.

[0054] The ultraviolet absorption spectra and fluorescence emission spectra of solution A and solution B were tested respectively.

[0055] UV absorption spectrum Figure 6 As shown in Figure A, it can be seen that after the addition of cysteine, the probe F-BTD shows a maximum absorption peak at 470 nm, and this wavelength is used as the optimal fluorescence excitation wavelength for subsequent fluorescence experiments.

[0056] Fluorescence emission spectra such as Figure 6 As shown in Figure B, it can be seen that after the probe F-BTD is added with cysteine, the fluorescence is enhanced under the excitation wavelength of 470nm and the maximum emission wavelength is 570nm, indicating the feasibility of F-BTD in detecting cysteine. x slit=20nm,E mslit=10nm, PMT=400V.

[0057] The pH of the buffer solution in solution A and solution B was changed respectively. The fluorescence emission intensity of probe F-BTD and the fluorescence intensity change curve after the reaction of probe F-BTD and cysteine ​​at pH 2 to 12 was as follows Figure 7 As shown in the figure, the probe F-BTD has excellent stability within the pH range investigated. In addition, the results also show that the optimal pH value for the probe F-BTD to respond to cysteine ​​is 7.4, further demonstrating the feasibility of the probe's application in biological systems.

[0058] The change of fluorescence emission intensity of solution B over time, such as Figure 8 As shown in the figure, the addition of cysteine ​​achieves a rapid response. Initially, the F-BTD probe exhibits virtually no fluorescence. However, after cysteine ​​addition, the fluorescence intensity increases over time, reaching a maximum around 120 seconds and then stabilizing. This demonstrates the potential of the F-BTD probe for rapid response and real-time monitoring of cysteine.

[0059] Example 3

[0060] Quantitative detection of cysteine ​​in solution using probe F-BTD

[0061] The concentration of cysteine ​​in solution B in Example 2 was replaced with a series of values, and the final concentrations of cysteine ​​in solution B were 0 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 100 μM, 150 μM, 200 μM, 300 μM, and 450 μM, respectively; then, the fluorescence emission spectrum of the sample was tested, and the testing conditions were the same as in Example 2.

[0062] like Figure 9 As shown in Figure A, it can be seen that the probe F-BTD has a good response to cysteine ​​in the concentration range of 0 to 450 μM; and as the cysteine ​​concentration increases, the fluorescence emission intensity of the system gradually increases, and there is a good linear relationship between the fluorescence emission intensity (y) of the system and the cysteine ​​concentration (x) in the range of 0 to 250 μM. Figure 9 As shown in B, the linear regression equation between the two is y = 7.7426x + 29.3016, R 2 = 0.9991. Furthermore, the calculated limit of detection (LOD) was 232 nM (S / N = 3) (LOD = 3б / k, where б is the standard deviation (SD) of eight blank solution measurements and k is the slope of the linear regression equation). These experimental results demonstrate that the probe F-BTD can quantitatively detect cysteine ​​within a certain concentration range in solution.

[0063] Example 3

[0064] Two-photon performance of the probe F-BTD

[0065] The final concentration of probe F-BTD in solution B in Example 2 was replaced with 43 μM, and the final concentration of cysteine ​​was 500 μM. At the same time, a 2 μM fluorescein at pH 11 was used as a reference, and a Leica TCS SP8 confocal scanning microscope and a multiphoton femtosecond laser were used as the excitation light source to record the fluorescence intensity at a wavelength of 680 to 880 nm. The two-photon absorption cross section (σ) of the probe F-BTD was calculated using the formula: Where the subscripts s and r represent the probe and reference, respectively, Φ is the fluorescence quantum yield, C is the concentration, n is the refractive index of the solvent, and F is the fluorescence intensity. Figure 10 As shown, the maximum two-photon absorption wavelength is 760 nm and the two-photon absorption cross section is 93 GM.

[0066] Example 4

[0067] Selectivity of probe F-BTD for cysteine

[0068] The cysteine ​​in the mixed solution B in Example 2 was replaced with other metal ions, amino acids, other biothiols, and reducing substances. The final concentrations of the analytes were 1. blank, 2.100 μM Val, 3.100 μM Trp, 4.100 μM Arg, 5.100 μM His, 6.100 μM Asp, 7.100 μM Glu, 8.1 mM Na + , 9.1mM K + 、10.1mM Ca 2 + 、11.1mM Mg 2+ 、12.100μM Zn 2+ 、13.100μM Cu 2+ 、14.100μM Fe 2+ 、15.100μM Fe 3+ , 16.100μM NaClO, 17.100μM NaNO2, 18.100μM H2O2, 19.5μM NaHSO3, 20.100μM Na2S, 21.100μM AA, 22.100μMCys; then the fluorescence emission intensity of each system was tested under excitation at a wavelength of 470nm.

[0069] like Figure 11 As shown in the figure, it can be seen that the probe F-BTD shows good specificity for cysteine ​​in the above analytes.

[0070] Example 5

[0071] Cytotoxicity of probe F-BTD

[0072] Cell culture: HeLa cells were cultured in RPMI1640 medium containing 10% fetal bovine serum, 1% 100 U / mL penicillin, and 100 μg / mL streptomycin in a biological incubator at 5% CO2 and 37°C.

[0073] Cytotoxicity experiment: Cytotoxicity was determined using the cell counting kit CCK-8 method. HeLa cells in logarithmic phase growth were cultured in 96-well plates. After adhesion, the cells were incubated with 0μM, 5μM, 10μM, 15μM, 20μM, 25μM, 30μM, 35μM, 40μM, and 45μM F-BTD for 24 hours. Then, 10μL of CCK-8 solution (5mg / mL) was added to each well and incubated for 1 hour. Finally, the absorbance at a wavelength of 450nm was measured using a microplate reader, and the cell viability was calculated based on the measured absorbance results. The results are shown in Figure 2. Figure 12 As shown, when the concentration of probe F-BTD is 45 μM, the survival rate of HeLa cells is still as high as over 80%. The results show that the probe F-BTD has good biocompatibility and can be used for fluorescence imaging of biological systems such as cells.

[0074] Example 6

[0075] Response mechanism of probe F-BTD to cysteine

[0076] Figure 13 This is the reaction mechanism of probe F-BTD and cysteine. Probe F-BTD uses dihydroxy-substituted 2,1,3-benzothiadiazole BTD-OH as the fluorescent matrix and 7-nitro-2,1,3-benzoxadiazole NBD-Cl as the recognition group, and the two are connected by an ether bond. Due to the strong electron-withdrawing ability of the recognition group NBD, the fluorescence of the probe F-BTD molecule itself is very weak. When the analyte cysteine ​​is added, due to the strong reducing ability of cysteine, the ether bond in the probe F-BTD breaks, releasing the fluorescent matrix BTD-OH. The mass spectrometry results of probe F-BTD and the reaction of probe F-BTD with cysteine ​​(such as Figure 14 , Figure 15 ) also confirmed Figure 13 The mass spectrometry data are as follows: HRMS of F-BTD before reaction: calcd 646.0655, found 646.0658; HRMS of BTD-OH, one of the products after the reaction of probe F-BTD and cysteine: calcd 320.0619, found 320.0620.

[0077] Example 7

[0078] Fluorescence imaging of endogenous and exogenous cysteine ​​in HeLa cells using the probe F-BTD

[0079] HeLa cells were selected to investigate the ability of the probe F-BTD to image endogenous and exogenous cysteine ​​fluorescence in cells. For exogenous cysteine ​​fluorescence imaging experiments, HeLa cells growing in logarithmic phase were cultured in 4-well cell culture dishes. After adhesion, the cells were divided into four groups. The first group, without additional cysteine, was selected as the control group, while the other three groups received cysteine ​​at concentrations of 50 μM, 150 μM, and 200 μM, respectively. To evaluate the ability of the probe F-BTD to image endogenous cysteine ​​fluorescence in HeLa cells, HeLa cells growing in logarithmic phase were divided into four groups. The first group was pretreated with 1 mM N-ethylmaleimide (NEM, a biothiol scavenger) for 30 minutes and then incubated with the probe F-BTD for 30 minutes. The second group was incubated with the probe F-BTD for 30 minutes. The third group was incubated with 200 μM cysteine ​​and the probe F-BTD for 30 minutes. The fourth group was pretreated with 1 mM NEM for 30 minutes and then incubated with 200 μM cysteine ​​and the probe F-BTD for 30 minutes. The concentration of the probe F-BTD was 10 μM. Before imaging, the cells were washed three times with phosphate-buffered saline. Fluorescence imaging of the cells was obtained by laser confocal microscopy in the red channel (550 nm to 600 nm) with 488 nm excitation.

[0080] Figure 16 This demonstrates the ability of probe F-BTD to image exogenous cysteine ​​fluorescence in HeLa cells; Figure 17 The results show that the probe F-BTD can image the fluorescence of endogenous cysteine ​​in HeLa cells.

[0081] Example 8

[0082] Two-photon fluorescence imaging of exogenous cysteine ​​in HeLa cells using probe F-BTD

[0083] HeLa cells were selected to investigate the ability of the probe F-BTD to image exogenous cysteine ​​with two-photon fluorescence. For the two-photon fluorescence imaging of exogenous cysteine, HeLa cells in logarithmic phase growth were cultured in 4-well cell culture dishes. After adhesion, the cells were divided into four groups. The first group, without additional cysteine, was selected as the control group. The other three groups received cysteine ​​at concentrations of 50 μM, 150 μM, and 200 μM, respectively. Two-photon fluorescence imaging of the cells was obtained using a laser confocal microscope in the red channel (550 nm to 600 nm) excited at 760 nm. Figure 18 The results demonstrated the ability of the probe F-BTD to image exogenous cysteine ​​in HeLa cells using two-photon fluorescence.

[0084] The above-mentioned reference examples provide a detailed description of a two-photon fluorescent probe based on 2,1,3-benzothiadiazole that rapidly responds to cysteine, its preparation method, and application. This description is illustrative rather than restrictive, and several embodiments may be listed according to the limited scope. Therefore, changes and modifications without departing from the overall concept of the present invention should fall within the scope of protection of the present invention.

Claims

1. A two-photon fluorescent probe based on 2,1,3-benzothiadiazole that rapidly responds to cysteine, characterized in that: The structural formula of the two-photon fluorescent probe is:

2. The method for preparing a two-photon fluorescent probe based on 2,1,3-benzothiadiazole fast-responding cysteine ​​according to claim 1, characterized in that: The preparation method comprises the following steps: (1) 4,7-dibromo-2,1,3-benzothiadiazole, 4-hydroxyphenylboronic acid pinacol ester, tetrabutylammonium bromide, and tetrakis(triphenylphosphine)palladium are added to a mixed solution of toluene and ethanol, and then a potassium carbonate solution is added. Under the protection of an inert gas, the mixture is stirred at 75 to 85° C. for 20 to 24 hours, and then the reaction solution is extracted, concentrated, purified, and dried to obtain a monohydroxy-substituted 2,1,3-benzothiadiazole compound BTD-Br. The structural formula of the BTD-Br is: (2) Substituting BTD-Br for 4,7-dibromo-2,1,3-benzothiadiazole in step (1), repeating step (1) to synthesize a dihydroxy-substituted 2,1,3-benzothiadiazole fluorescent matrix BTD-OH, wherein the structural formula of BTD-OH is: (3) BTD-OH and 4-chloro-7-nitro-2,1,3-benzothiadiazole NBD-Cl were dissolved in anhydrous acetonitrile, and then potassium carbonate was added and stirred at room temperature for 6 to 8 hours. After the reaction was completed, the mixture was centrifuged, washed, and dried to obtain the 2,1,3-benzothiadiazole-based fast-response cysteine ​​two-photon fluorescent probe.

3. The preparation method according to claim 2, characterized in that In step (1), the molar ratio of 4,7-dibromo-2,1,3-benzothiadiazole, 4-hydroxyphenylboronic acid pinacol, tetrabutylammonium bromide, and tetrakis(triphenylphosphine)palladium is 1:1.2-1.5:0.05-0.06:0.01-0.

02.

4. The preparation method according to claim 2, characterized in that In step (1), the volume ratio of toluene to ethanol is 3:2; the concentration of 4,7-dibromo-2,1,3-benzothiadiazole in the mixed solution of toluene and ethanol is 0.1-0.2 mol / L.

5. The preparation method according to claim 2, characterized in that In step (1), the concentration of the potassium carbonate solution is 1.5 to 2.5 mol / L; and the volume ratio of the potassium carbonate solution to the mixed solution of toluene and ethanol is 1:

5.

6. The preparation method according to claim 2, characterized in that In step (1), the purification method is: using ethyl acetate: petroleum ether = 1:3-5 as eluent, purifying the product through a silica gel column, collecting the product and concentrating it.

7. The preparation method according to claim 2, characterized in that In step (3), the molar ratio of BTD-OH, NBD-Cl and potassium carbonate is 1:2.2-2.5:3-5.

8. The preparation method according to claim 2, characterized in that In step (3), the concentration of NBD-Cl in the acetonitrile solution is 0.01 to 0.02 mol / L.

9. Use of the 2,1,3-benzothiadiazole-based two-photon fluorescent probe that rapidly responds to cysteine ​​as claimed in claim 1 in the quantitative detection of cysteine ​​for purposes other than disease diagnosis and treatment.

10. Use of the 2,1,3-benzothiadiazole fast-response cysteine ​​two-photon fluorescent probe according to claim 1 in HeLa cell fluorescence imaging for purposes other than disease diagnosis and treatment.

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