CN117186088A - Two-photon fluorescent probe based on 2,1, 3-benzothiadiazole rapid response cysteine and preparation method and application thereof - Google Patents
Two-photon fluorescent probe based on 2,1, 3-benzothiadiazole rapid response cysteine and preparation method and application thereof Download PDFInfo
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a two-photon fluorescent probe based on 2,1, 3-benzothiadiazole quick response cysteine, a preparation method and application thereof, and the two-photon fluorescent probe F-BTD based on 2,1, 3-benzothiadiazole quick response cysteine is synthesized through three steps, has two-photon performance, can be used for quickly and sensitively quantitatively detecting cysteine, can carry out fluorescent imaging on exogenous and endogenous cysteines of HeLa cells, and has the capacity of carrying out two-photon fluorescent imaging on exogenous cysteines in cells.
Description
Technical Field
The invention belongs to the field of small molecular fluorescent probes, and particularly relates to a two-photon fluorescent probe based on 2,1, 3-benzothiadiazole rapid response cysteine, and a preparation method and application thereof.
Background
Cysteine is an important endogenous thiol in organisms and is considered an important biomolecule for maintaining redox homeostasis. The intracellular concentration of cysteine varies between 30-200. Mu.M, with a total plasma concentration of about 250. Mu.M. Under normal conditions, the production, consumption, aggregation and elimination of cysteine in the life system are in a dynamic balance state, and the fluctuation of concentration is related to various diseases, including neurodegenerative diseases such as Alzheimer disease, parkinson disease and the like, cardiovascular diseases, liver injury, epilepsy and the like. Therefore, there is a need to monitor and quantify the concentration levels of cysteine in biological systems to assess their distribution and metabolic profile, which is critical to understanding the role of cysteine in the chemical and biological fields.
In recent years, methods such as high performance liquid chromatography, mass spectrometry, capillary zone electrophoresis and the like are widely applied to measurement of cysteine, and particularly, fluorescence detection of cysteine has been rapidly developed. More importantly, the cysteine-responsive fluorescent probe has strong analysis and identification capabilities, and improves the sensitivity and speed of detection. Furthermore, fluorescent probes have been considered as powerful tools for measuring cysteine due to their high spatial-temporal resolution and sensitivity, especially in vivo analysis. To date, most cysteine-responsive fluorescent probes are based on the redox and nucleophilicity of cysteine. The adopted fluorescence sensing mechanism mainly comprises acrylate cyclization, aldehyde cyclization, michael addition reaction and the like. The currently reported fluorescent precursors that respond to cysteine are mainly focused on coumarin, quinoline, naphthalimide, fluorescein, and BODIPY. Thus, there is a continuing need to use new fluorescent precursors to detect cysteines to increase imaging depth and signal to noise ratio of biological systems.
Disclosure of Invention
The invention aims to provide a two-photon fluorescent probe based on 2,1, 3-benzothiadiazole fast response cysteine and a preparation method thereof, wherein the two-photon fluorescent probe has the advantages of high reaction speed, strong specificity, good sensitivity and excellent two-photon performance.
The invention also aims to provide the application of the two-photon fluorescent probe based on the 2,1, 3-benzothiadiazole rapid response cysteine in quantitative detection of the cysteine, wherein the sensitivity is high and the detection limit is as low as 232nM when the two-photon fluorescent probe is used for quantitatively detecting the concentration of the cysteine.
The invention also aims to provide the application of the two-photon fluorescent probe based on the 2,1, 3-benzothiadiazole fast response cysteine in cell fluorescence imaging for non-disease diagnosis and treatment purposes, and the two-photon fluorescent probe can realize fluorescence imaging of endogenous and exogenous cysteines of HeLa cells and two-photon fluorescence imaging of exogenous cysteines of HeLa cells.
The technical scheme adopted by the invention for achieving the purpose is as follows:
the invention provides a two-photon fluorescent probe based on 2,1, 3-benzothiadiazole fast response cysteine, which is characterized in that the structural formula of the two-photon fluorescent probe is as follows:
the invention also provides a preparation method of the two-photon fluorescent probe based on the 2,1, 3-benzothiadiazole rapid response cysteine, which comprises the following steps:
(1) Adding 4, 7-dibromo-2, 1, 3-benzothiadiazole, 4-hydroxyphenylboronic acid pinacol ester, tetrabutylammonium bromide and tetrakis (triphenylphosphine) palladium into a toluene and ethanol mixed solution, then adding a potassium carbonate solution, stirring and reacting for 20-24 hours at 75-85 ℃ under the protection of inert gas, and then extracting, concentrating, purifying and drying the reaction liquid to obtain a monohydroxy substituted 2,1, 3-benzothiadiazole compound BTD-Br, wherein the structural formula of the BTD-Br is as follows:
(2) Replacing 4, 7-dibromo-2, 1, 3-benzothiadiazole in the step (1) with BTD-Br, and repeating the step (1) to synthesize a dihydroxyl substituted 2,1, 3-benzothiadiazole fluorescent parent BTD-OH, wherein the structural formula of the BTD-OH is as follows:
(3) Dissolving BTD-OH and 4-chloro-7-nitro-2, 1, 3-benzoxadiazole (NBD-Cl) in anhydrous acetonitrile, then adding potassium carbonate, stirring for 6-8 h at room temperature, centrifuging, washing and drying after the reaction is finished, thus obtaining the two-photon fluorescent probe F-BTD based on 2,1, 3-benzothiadiazole fast response cysteine. In the step (1), the mass ratio of the substances 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.
In the step (1), the volume ratio of toluene to ethanol is 3:2; the concentration of the 4, 7-dibromo-2, 1, 3-benzothiadiazole in the mixed solution of toluene and ethanol is 0.1-0.2 mol/L.
In the step (1), the concentration of the potassium carbonate solution is 1.5-2.5 mol/L; the volume ratio of the potassium carbonate solution to the toluene and ethanol mixed solution is 1:5.
In the step (1), the purification method comprises the following steps: ethyl acetate: petroleum ether = 1: 3-5 is eluent, and the product is collected by purification of a silica gel column and concentrated.
In the step (2), the ratio of the amounts of substances of BTD-Br, 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.
In the step (2), the purification method of the crude product comprises the following steps: ethyl acetate: petroleum ether = 1: 2-4 is eluent, and the product is collected by purification of a silica gel column and concentrated.
In the step (3), the ratio of the amounts of BTD-OH, NBD-Cl and potassium carbonate is 1:2.2 to 2.5:3 to 5.
In the step (3), the concentration of NBD-Cl in the acetonitrile solution is 0.01-0.02 mol/L.
The invention also provides application of the two-photon fluorescent probe based on the 2,1, 3-benzothiadiazole rapid response cysteine in quantitative detection of the cysteine.
The invention also provides application of the two-photon fluorescent probe based on the 2,1, 3-benzothiadiazole rapid response cysteine in cytofluorescence imaging for non-disease diagnosis and treatment purposes.
The invention adopts a three-step synthesis method to simply and conveniently synthesize the fluorescent probe F-BTD based on 2,1, 3-benzothiadiazole quick response cysteine, wherein in the structure, dihydroxyl substituted 2,1, 3-benzothiadiazole is a fluorophore, and nitro-2, 1, 3-benzoxadiazole (NBD-Cl) is an identification group, and the two are connected through ether bonds. The recognition group NBD in the structure has strong electron withdrawing capability, so that the probe molecule F-BTD has weak fluorescence. However, when cysteine is added, the ether bond is broken due to the strong reducing capability of the cysteine, so that a fluorescent parent is released, and the fluorescence of the system is enhanced.
The in vitro experiment result shows that F-BTD can respond to cysteine within 2min and has two-photon performance, and the two-photon absorption cross section is 93GM. Second, F-BTD has good specificity, and the probe has no obvious response to metal ions, amino acids, other biological thiols and reducing substances in biological systems. Cell imaging experiment results show that F-BTD can perform fluorescence imaging on endogenous and exogenous cysteine of HeLa cells. More importantly, the fluorescent imaging device can carry out two-photon fluorescence imaging on exogenous cysteine of HeLa cells. Therefore, F-BTD has good application prospect in cysteine related research and monitoring.
Compared with the prior art, the two-photon fluorescent probe F-BTD based on the 2,1, 3-benzothiadiazole fast response cysteine has the advantages of high reaction speed, strong specificity, good sensitivity and excellent two-photon performance.
Drawings
FIG. 1 is a structural formula of a probe F-BTD;
FIG. 2 is a synthetic route diagram of probe F-BTD;
FIG. 3 is a nuclear magnetic hydrogen spectrum of BTD-Br;
FIG. 4 is a nuclear magnetic hydrogen spectrum of BTD-OH;
FIG. 5 is a nuclear magnetic resonance hydrogen spectrum of probe F-BTD;
FIG. 6 is a graph of ultraviolet absorption spectrum (A) and a graph of fluorescence emission spectrum (B) of the probe F-BTD, the probe F-BTD and cysteine (Cys) of example 2 after reaction;
FIG. 7 is a graph showing the change in fluorescence emission intensity with pH after the reaction of the probe F-BTD, the probe F-BTD and cysteine (Cys) in example 2, wherein the pH value ranges from 2 to 12;
FIG. 8 is a graph showing the relationship between fluorescence emission intensity and time after the reaction of the probe F-BTD and cysteine (Cys) in example 2;
FIG. 9 is a graph (A) of fluorescence emission spectrum and a graph (B) of the linear relationship between fluorescence emission intensity and cysteine (Cys) concentration after the reaction of the probe F-BTD with cysteine (Cys) at different concentrations;
FIG. 10 is a two-photon performance diagram of probe F-BTD;
FIG. 11 shows the relationship between the fluorescence intensity of the probe F-BTD specifically recognizing cysteine (Cys) and the difference between the blank and the blank, wherein the analytes are 1. Control, 2.Val, 3.Trp, 4.Arg, 5.His, 6.Asp, 7.Glu, 8.Na, respectively + 、9.K + 、10.Ca 2+ 、11.Mg 2+ 、12.Zn 2+ 、13.Cu 2+ 、14.Fe 2+ 、15.Fe 3+ 、16.NaClO、17.NaNO 2 、18.H 2 O 2 、19.NaHSO 3 、20.Na 2 S、21.AA、22.Cys；
FIG. 12 is cytotoxicity of probe F-BTD;
FIG. 13 is a mechanism diagram of probe F-BTD response to cysteine (Cys);
FIG. 14 is a mass spectrum of probe F-BTD;
FIG. 15 is a mass spectrum of probe F-BTD after reaction with cysteine (Cys);
FIG. 16 is a fluorescence imaging of F-BTD probe to HeLa cell exogenous cysteine (Cys);
FIG. 17 is a fluorescent imaging of probe F-BTD on HeLa cells endogenous cysteine (Cys); FIG. 18 is a two-photon fluorescence imaging of F-BTD probe to HeLa cell exogenous cysteine (Cys).
Detailed Description
Example 1
A two-photon fluorescent probe based on 2,1, 3-benzothiadiazole fast response cysteine has a structural formula:
the synthetic route of the cysteine-responsive fluorescent probe based on the 2,1, 3-benzothiadiazole as a matrix is shown in figure 2, and the synthetic method comprises the following steps:
(1) Synthetic compoundsBTD-Br: 0.5830g (2 mmoL) of 4, 7-dibromo-2, 1, 3-benzothiadiazole and 0.572g (2.6 mmoL) of 4-hydroxyphenylboronic acid pinacol ester are weighed, 0.0310g (0.1 mmoL) of tetrabutylammonium bromide and 0.0392g (0.03 mmoL) of tetrakis (triphenylphosphine) palladium are then weighed, 9mL of toluene and 6mL of absolute ethyl alcohol are added, and finally 3mL of a potassium carbonate solution with the concentration of 2mol/L (0.6415 g) is added, the mixture is uniformly mixed, the air in the round bottom flask is removed, and the mixture is refluxed at 75 ℃ for 24 hours under the protection of nitrogen atmosphere. After the completion of the reaction, the reaction was cooled to room temperature naturally and rapidly poured into 100mL of water to quench the reaction, followed by extraction three times with 50mL of methylene chloride, washing three times with 50mL of water and three times with 50mL of saturated brine. After drying over anhydrous magnesium sulfate, the dichloromethane was removed by rotary evaporation, and purified by silica gel column with ethyl acetate as eluent: petroleum ether = 1:4 (V: V). Vacuum drying to obtain intermediate BTD-Br, whose nuclear magnetic hydrogen spectrum is shown in figure 3, 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)。
(2) Synthesis of Compound BTD-OH: the synthesis step and the pretreatment method are the same as in step 1, except that 4, 7-dibromo-2, 1, 3-benzothiadiazole in step (1) is replaced by BTD-Br in the same amount; the purification method comprises the following steps: purifying by silica gel column, eluting with ethyl acetate: petroleum ether = 1:2 (V/V). Vacuum drying to obtain fluorescent parent BTD-OH, whose nuclear magnetic hydrogen spectrum is shown in figure 4, 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)。
(3) Fluorescent probe F-BTD synthesis: 0.0141g (0.044 mmol) of BTD-OH and 0.0183g (0.098 mmol) of 4-chloro-7-nitro-2, 1, 3-benzoxadiazole (NBD-Cl) were weighed into 7mL of anhydrous acetonitrile, and 0.0195g (0.141 mmol) of catalyst potassium carbonate was added and stirred at room temperature for 6 hours. After the reaction was completed, the mixture was centrifuged at 8000rpm for 10 minutes to obtain a precipitate. And washing the precipitate with water for three times, and freeze-drying to obtain the two-photon fluorescent probe F-BTD based on the 2,1, 3-benzothiadiazole fast response cysteine. The nuclear magnetic hydrogen spectrum is shown in figure 5. 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)。
Example 2
Response properties of probe F-BTD to cysteine
20. Mu.L of a probe F-BTD dimethyl sulfoxide solution with a concentration of 500. Mu.M was added to 980. Mu.L of a phosphate buffer solution with a concentration of 20mM and a pH of 7.40 to obtain a solution A;
taking 20 mu L of a dimethyl sulfoxide solution of a probe F-BTD with the concentration of 500 mu M, adding 930 mu L of a phosphate buffer solution with the concentration of 20mM and the pH of 7.40, and then adding 50 mu L of a cysteine solution with the concentration of 10mM to obtain a solution B, wherein the final concentration of cysteine in the solution B is 500 mu M;
the ultraviolet absorption spectrum and the fluorescence emission spectrum of the solution a and the solution B were respectively tested.
The ultraviolet absorption spectrum is shown in FIG. 6A, and it can be seen from the graph that a maximum absorption peak appears at 470nm after the probe F-BTD is added with cysteine, and the wavelength is used as the optimal fluorescence excitation wavelength for the subsequent fluorescence experiment.
The fluorescence emission spectrum is shown in FIG. 6B, and it can be seen from the graph that the fluorescence of the probe F-BTD under the excitation of 470nm is enhanced and the maximum emission wavelength is 570nm after the cysteine is added, so that the feasibility of detecting the cysteine by the F-BTD is shown. Fluorescent test conditions E x slit＝20nm，E m slit＝10nm，PMT＝400V。
The pH of the buffer solution in the solution A and the buffer solution in the solution B are respectively changed, and the change curves of fluorescence emission intensities of the probe F-BTD, the probe F-BTD and cysteine after the reaction at pH 2-12 are shown in FIG. 7. As can be seen from the figure, the probe F-BTD has excellent stability in the pH range under investigation; in addition, the results also show that the optimal pH of the probe F-BTD responsive to cysteine is 7.4, further illustrating the possibility of probe application in biological systems.
The change of fluorescence emission intensity of solution B with time is shown in FIG. 8, and it can be seen from the graph that the effect of rapid response can be achieved after adding cysteine. Upon addition, probe F-BTD was essentially non-fluorescent, and after cysteine addition, the fluorescence intensity increased over time and reached a maximum at 120s and tended to stabilize, indicating the potential of probe F-BTD to rapidly respond, monitoring cysteine in real time.
Example 3
Quantitative detection of cysteine in solution by using F-BTD probe
The concentration of cysteine in solution B in example 2 was replaced with a range of values, the final concentration of cysteine in solution B being 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,450 μm, respectively; the samples were then tested for fluorescence emission spectra under the same conditions as in example 2.
As shown in FIG. 9A, it can be seen that the probe F-BTD has a good response to cysteine at a concentration ranging from 0 to 450. Mu.M; and the fluorescence emission intensity of the system is gradually increased along with the increase of the concentration of the cysteine, and a good linear relation exists between the fluorescence emission intensity (y) of the system and the concentration (x) of the cysteine in the range of 0-250 mu M. As shown in fig. 9B, the linear regression equation between the two is y=7.7426x+29.3016, r 2 = 0.9991. In addition, the lowest detection Limit (LOD) was calculated to be 232nM (S/n=3) (lod=3δ/k, δ being the standard deviation SD of measuring eight blank solutions, k being the slope of the linear regression equation). The experimental result shows that the probe F-BTD can quantitatively detect cysteine with a certain concentration range in the solution.
Example 3
Two-photon Performance of Probe F-BTD
The final concentration of probe F-BTD in solution B of example 2 was replaced with 43. Mu.M and the final concentration of cysteine was 500. Mu.M. Meanwhile, using fluorescein with ph=11, 2 μm as a reference, a Leica TCS SP8 confocal scanning microscope and a multiphoton femtosecond laser as excitation light sources and recording fluorescence intensities at wavelengths of 680 to 880nm, and calculating a two-photon absorption cross section (σ) of the probe F-BTD using a formula. The formula isWherein subscripts s and r represent the probe and reference, respectively, the letter Φ is the fluorescence quantum yield, C is the concentration, n is the refractive index of the solvent, and F is the fluorescence intensity. As shown in FIG. 10, the two-photon maximum absorption wavelength was 760nm, and the two-photon absorption cross section was 93GM.
Example 4
Selectivity of probe F-BTD for cysteine
The cysteine in the mixed solution B in example 2 was replaced with other metal ions, amino acids, other biological thiols and reducing substances, respectively. The final concentrations of each analyte were 1.blank, 2.100. Mu.MVal, 3.100. Mu.M Trp, 4.100. Mu.M Arg, 5.100. Mu.M His, 6.100. Mu.M Asp, 7.100. Mu.M Glu, 8.1mM Na, respectively + 、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 NaNO 2 、18.100μM H 2 O 2 、19.5μM NaHSO 3 、20.100μM Na 2 S, 21.100. Mu.M AA, 22.100. Mu.MCys; each system was then tested for fluorescence emission intensity under 470nm wavelength excitation.
As shown in FIG. 11, it can be seen that the probe F-BTD exhibits good specificity for cysteine in the above-mentioned analytes.
Example 5
Cytotoxicity of Probe F-BTD
Cell culture: heLa cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 1%100U/mL penicillin and 100. Mu.g/mL streptomycin. The culture condition of the biological incubator is 5% CO 2 ，37℃。
Cytotoxicity experiment: cytotoxicity was determined using the cell counting kit CCK-8 method. HeLa cells grown in log phase were cultured in 96-well plates. After adhesion, cells were incubated with 0. Mu.M, 5. Mu.M, 10. Mu.M, 15. Mu.M, 20. Mu.M, 25. Mu.M, 30. Mu.M, 35. Mu.M, 40. Mu.M, 45. Mu. M F-BTD for 24 hours. Then, 10. Mu.L of CCK-8 solution (5 mg/mL) was added to each well and incubated for 1 hour. Finally, the absorbance at a wavelength of 450nm was measured with a microplate reader, and the cell viability was calculated based on the measured absorbance results. As shown in FIG. 12, the HeLa cell viability is still up to 80% or more at a probe F-BTD concentration of 45. Mu.M, and the results indicate that the probe F-BTD has good biocompatibility and can be used for fluorescence imaging of biological systems such as cells.
Example 6
Mechanism of response of probe F-BTD to cysteine
FIG. 13 shows the reaction mechanism of the probe F-BTD with cysteine. The probe F-BTD takes dihydroxyl substituted 2,1, 3-benzothiadiazole BTD-OH as a fluorescent parent body, and 7-nitro-2, 1, 3-benzoxadiazole NBD-Cl as a recognition group, and the two are connected by ether bonds. The probe F-BTD molecule itself is very fluorescent due to the strong electron withdrawing ability of the recognition group NBD. After the cysteine to be detected is added, the ether bond in the probe F-BTD is broken due to the strong reducing capability of the cysteine, so that the fluorescent parent BTD-OH is released. The mass spectrum results (FIG. 14, FIG. 15) of the probe F-BTD and the probe F-BTD after the reaction with cysteine also confirm the reaction mechanism in FIG. 13. Mass spectrum data are respectively: HRMS of pre-reaction F-BTD: calcd 646.0655, found646.0658; HRMS of one of the products BTD-OH after probe F-BTD and cysteine reaction: calcd320.0619; found 320.0620.
Example 7
Fluorescent imaging of F-BTD probe on endogenous and exogenous cysteines of HeLa cells
HeLa cells were selected to investigate the ability of probe F-BTD to fluorescent image intracellular exogenous and endogenous cysteines. For exogenous cysteine fluorescence imaging experiments, heLa cells grown in log 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 a control group, and the other three groups were added with cysteine at concentrations of 50. Mu.M, 150. Mu.M, 200. Mu.M, respectively. To assess the ability of probe F-BTD to fluorescent image of endogenous cysteines in HeLa cells, heLa cells grown in log phase were divided into four groups. Pretreatment with 1mM N-ethylmaleimide (NEM, a biological thiol scavenger) for 30 min for the first group followed by incubation with probe F-BTD for 30 min; the second group incubated HeLa cells with probe F-BTD for 30 minutes; the third group incubated HeLa cells with 200. Mu.M cysteine and probe F-BTD for 30 min; fourth, heLa cells were first pretreated with 1mM NEM for 30 min, then incubated with 200. Mu.M cysteine and probe F-BTD for 30 min. Wherein the concentration of the probe F-BTD is 10. Mu.M. Prior to imaging, cells were washed three times with phosphate buffer solution. Fluorescence imaging of cells was obtained by laser confocal microscopy in 488nm excited red channel (550 nm-600 nm).
FIG. 16 shows the ability of probe F-BTD to fluorescent image HeLa cells exogenous cysteine; FIG. 17 shows the ability of probe F-BTD to fluorescent image of endogenous cysteine in HeLa cells.
Example 8
Two-photon fluorescence imaging of exogenous cysteine of HeLa cells by using F-BTD (probe-based fluorescence)
HeLa cells were selected to investigate the ability of probe F-BTD to image intracellular exogenous cysteine two-photon fluorescence. For exogenous cysteine two-photon fluorescence imaging experiments, heLa cells grown in log 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 a control group, and the other three groups were added with cysteine at concentrations of 50. Mu.M, 150. Mu.M, 200. Mu.M, respectively. Two-photon fluorescence imaging of cells was obtained in 760nm excited red channel (550 nm-600 nm) by laser confocal microscopy. The results of FIG. 18 demonstrate the ability of probe F-BTD to image exogenous cysteine two-photon fluorescence in HeLa cells.
The foregoing detailed description of a 2,1, 3-benzothiadiazole rapid-response cysteine-based two-photon fluorescent probe, and a method for preparing and using the same, with reference to examples, is illustrative and not restrictive, and several examples can be enumerated according to the scope of the present invention, and therefore, variations and modifications without departing from the general inventive concept should fall within the scope of protection of the present invention.
Claims (10)
1. The two-photon fluorescent probe based on the 2,1, 3-benzothiadiazole fast response cysteine is characterized by comprising the following structural formula:
2. the method for preparing the 2,1, 3-benzothiadiazole fast response cysteine-based two-photon fluorescent probe according to claim 1, wherein the preparation method comprises the following steps:
(1) Adding 4, 7-dibromo-2, 1, 3-benzothiadiazole, 4-hydroxyphenylboronic acid pinacol ester, tetrabutylammonium bromide and tetrakis (triphenylphosphine) palladium into a toluene and ethanol mixed solution, then adding a potassium carbonate solution, stirring and reacting for 20-24 hours at 75-85 ℃ under the protection of inert gas, and then extracting, concentrating, purifying and drying the reaction liquid to obtain a monohydroxy substituted 2,1, 3-benzothiadiazole compound BTD-Br, wherein the structural formula of the BTD-Br is as follows:
(2) Replacing 4, 7-dibromo-2, 1, 3-benzothiadiazole in the step (1) with BTD-Br, and repeating the step (1) to synthesize a dihydroxyl substituted 2,1, 3-benzothiadiazole fluorescent parent BTD-OH, wherein the structural formula of the BTD-OH is as follows:
(3) Dissolving BTD-OH and 4-chloro-7-nitro-2, 1, 3-benzoxadiazole (NBD-Cl) in anhydrous acetonitrile, then adding potassium carbonate, stirring for 6-8 h at room temperature, centrifuging, washing and drying after the reaction is finished, thus obtaining the two-photon fluorescent probe F-BTD based on 2,1, 3-benzothiadiazole fast response cysteine.
3. The method according to claim 2, wherein in the step (1), the ratio of the amounts of the substances 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.
4. The method according to claim 2, wherein in the step (1), the volume ratio of toluene to ethanol is 3:2; the concentration of the 4, 7-dibromo-2, 1, 3-benzothiadiazole in the mixed solution of toluene and ethanol is 0.1-0.2 mol/L.
5. The method according to claim 2, wherein in the step (1), the concentration of the potassium carbonate solution is 1.5 to 2.5mol/L; the volume ratio of the potassium carbonate solution to the toluene and ethanol mixed solution is 1:5.
6. The method of claim 2, wherein in step (1), the purification method is: ethyl acetate: petroleum ether = 1: 3-5 is eluent, and the product is collected by purification of a silica gel column and concentrated.
7. The method according to claim 2, wherein in the step (3), the ratio of the amounts of the substances BTD-OH, NBD-Cl, and potassium carbonate is 1:2.2 to 2.5:3 to 5.
8. The process according to claim 2, wherein in the step (3), the concentration of NBD-Cl in acetonitrile solution is 0.01 to 0.02mol/L.
9. The use of a two-photon fluorescent probe based on 2,1, 3-benzothiadiazole fast response cysteine as claimed in claim 1 for quantitative detection of cysteine.
10. Use of a two-photon fluorescent probe based on 2,1, 3-benzothiadiazole in rapid response cysteine as claimed in claim 1 for cytofluorescence imaging for non-disease diagnosis and treatment purposes.
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