CN114736199B - Methylene blue-based near-infrared fluorescent probe and synthetic method and application thereof - Google Patents
Methylene blue-based near-infrared fluorescent probe and synthetic method and application thereof Download PDFInfo
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Abstract
The invention provides a method for preparing a product based onA methyl blue near-infrared fluorescent probe and a synthetic method and application thereof. The probe compound is named 4- ((7- (diethylamino) -3-formyl-2-oxo-2H-chromium-4-yl) oxy) benzyl 3,7-bis (dimethylamino) -10H-phenothiazin-10-carboxylate, and the English name is 4- ((7- (diethylamino) -3-formyl-2-oxo-2H-chromen-4-yl) oxy) benzyl-3,7-bis (dimethylamino) -10H-phenothizine-10-carboxylate. The invention also provides a tracing method capable of detecting the metabolic process of GSH, namely the probe is applied to living cell imaging, and the GSH can be metabolized into SO in the living cells through a laser scanning confocal microscope 2 The process of (2) is dynamically traced. The detection method has the advantages of high sensitivity, simple method and simple operation.
Description
Technical Field
The invention relates to a methylene blue-based near-infrared fluorescent probe, in particular to a methylene blue-based near-infrared fluorescent probe, a synthetic method thereof and application of the probe in distinguishing and detecting mercaptan and metabolism.
Background
Metabolism is the general term for a series of ordered chemical reactions that maintain life in the body. Metabolism is also referred to as cellular metabolism. Chemical biology needs to develop new techniques and methods to study important molecular events in the signal transduction process of living systems using chemical small molecules as probes. With the improvement of organic synthesis technology, the high-performance multifunctional fluorescent probe based on organic molecules is prepared, and can be effectively used as a visual fluorescent tracing of a signal path of a living system and a medicine for effectively intervening physiological and pathological processes.
There is an important class of sulfur-containing active substances in the living system, mainly three kinds of biological thiols including homocysteine (HCy), cysteine (Cys), glutathione (GSH), sulfur dioxide (SO) 2 ) And hydrogen sulfide (H) 2 S). They form an interwoven network in the organism: cys is involved in the synthesis of various proteins, and the major antioxidant, GSH, is also a central substance of sulfur metabolism in cells. In recent years, studies have shown that the concentration and metabolic abnormalities of active sulfur-containing molecules are closely related to various diseases. Biological mercaptans and hydrogen sulfide are closely related to cardiovascular disease. Endogenous sulfur dioxide concentrations are closely related to the development and progression of cancer. The research on the metabolic pathway is beneficial toEffective intervention to prevent the occurrence and development of related major diseases.
In recent years, our research groups have focused on the chemically accurate measurement and bioimaging of sulfur-containing active species based on organic molecular fluorescent probes. In particular, advances have been made in the application of multi-site reactions and multi-color emission to signal paths.
In the invention, reaction sites are constructed at the same time to further distinguish targets or detect metabolites thereof, thereby realizing the simultaneous visual tracing of various metabolic pathways. Realizes the distinguishing detection of three kinds of biological mercaptan and the tracing of GSH metabolism SO 2 Detection of (3).
Disclosure of Invention
The invention aims to provide a methylene blue-based near-infrared fluorescent probe, a synthesis method thereof and application of the probe in distinguishing and detecting mercaptan and metabolism.
The invention provides a near-infrared fluorescent probe based on methylene blue, which is named as 4- ((7- (diethylamino) -3-formyl-2-oxo-2H-chromium-4-yl) oxy) benzyl 3,7-bis (dimethylamino) -10H-phenothiazine-10-carboxylate 4- ((7- (dimethylamino) -3-methyl-2-oxo-2H-chromen-4-yl) oxy) benzyl 3,7-bis (dimethylamino) -10H-phenothiazine-10-carboxylate. Designated MB-C, having the following structural formula:
the invention provides a method for synthesizing a methylene blue-based near-infrared fluorescent probe, which comprises the following steps:
1) Slowly adding phosphorus oxychloride into a mixture of malonic acid and phenol at 0 ℃, heating the mixture at 115 ℃ until the strong release of hydrogen chloride gas stops (about 1.5 hours), pouring the upper layer into a proper amount of water, extracting with ethyl acetate for 3 times, drying an organic phase with anhydrous sodium sulfate, and spin-drying the solvent to obtain pale yellow oily diphenyl malonate; wherein the feeding molar ratio is that the molar ratio of the malonic acid to the phenol to the phosphorus oxychloride = 1.2-1.5;
2) Dissolving diphenyl malonate in toluene, adding 3-diethylaminophenol, performing reflux reaction for 7 hours, performing suction filtration, washing with petroleum ether or n-hexane, and performing vacuum drying on a product to obtain a light yellow solid 7- (diethylamino) -4-hydroxy-2H-chromene-2-one; wherein the feeding molar ratio is that the diphenyl malonate is 3-diethylaminophenol = 1;
3) Adding N, N-dimethylformamide into phosphorus oxychloride in an ice-water bath, stirring for half an hour to obtain a transparent viscous substance, and dropwise adding 7- (diethylamino) -4-hydroxy-2H-chromene-2-one dissolved in N, N-dimethylformamide into the solution to obtain a castoreum solution; stirring for 12 hours at 60 ℃, then pouring into ice water, adding sodium hydroxide to adjust the pH value to 5 to generate a large amount of precipitate, carrying out suction filtration, washing with water, and then recrystallizing with absolute ethyl alcohol to obtain 4-chloro-7- (diethylamino) -2-oxo-2H-chromene-3-formaldehyde; wherein the feeding molar ratio of N, N-dimethylformamide to phosphorus oxychloride to 7- (diethylamino) -4-hydroxy-2H-chromen-2-one = 3-3.5;
4) Dissolving 4-chloro-7- (diethylamino) -2-oxo-2H-chromene-3-formaldehyde in dichloromethane, stirring for 5 minutes in an ice water bath, dropwise adding triethylamine, continuously stirring for 10 minutes, adding p-hydroxybenzyl alcohol, stirring for reacting for 8 hours, removing the solvent, and purifying the crude product by silica gel column chromatography to obtain a yellow product, namely 7- (diethylamino) -4- (4- (hydroxymethyl) phenoxy) -2-oxo-2H-chromene-3-formaldehyde; wherein the feeding molar ratio is 4-chloro-7- (diethylamino) -2-oxo-2H-chromene-3-formaldehyde to triethylamine to p-hydroxybenzyl alcohol = 3-3.5;
5) According to the mol ratio of 1.2-1.5:1-1.2:3-3.5:1-1.2 dissolving 7- (diethylamino) -4- (4- (hydroxymethyl) phenoxy) -2-oxo-2H-chromene-3-formaldehyde, 3,7-bis (dimethylamino) -10H-phenothiazine-10-carbonyl chloride, sodium carbonate and 4-dimethylaminopyridine in dichloromethane, and stirring for reaction for 8 hours under the conditions of nitrogen protection and ice bath; the solvent was removed and the crude product was purified by silica gel column chromatography to give 4- ((7- (diethylamino) -3-formyl-2-oxo-2H-chromen-4-yl) oxy) benzyl 3,7-bis (dimethylamino) -10H-phenothiazin-10-carboxylate as a yellow solid, named MB-C.
Preferably, the method comprises the following steps: in the step 1), the feeding molar ratio of malonic acid to phenol to phosphorus oxychloride =1: 1.2; in the step 2), the feeding molar ratio of diphenyl malonate to 3-diethylaminophenol = 1. In the step 3), the feeding molar ratio of N, N-dimethylformamide to phosphorus oxychloride to 7- (diethylamino) -4-hydroxy-2H-chromen-2-one = 3. In the step 4), the feeding molar ratio of 4-chloro-7- (diethylamino) -2-oxo-2H-chromene-3-formaldehyde to triethylamine to p-hydroxybenzol = 3. The molar ratio of 7- (diethylamino) -4- (4- (hydroxymethyl) phenoxy) -2-oxo-2H-chromene-3-carbaldehyde, 3,7-bis (dimethylamino) -10H-phenothiazine-10-carbonyl chloride, sodium carbonate, and 4-dimethylaminopyridine in step 5) was 1.2:1:3:1.
the near-infrared fluorescent probe MB-C can be used for distinguishing and detecting Cys, hcy and GSH and metabolizing SO by GSH 2 Detection of (3). The detection principle is as follows:
the probe takes oxygen on hydroxyl of a connecting group p-hydroxybenzyl alcohol as reaction sites of three thiols, one part releases methylene blue fluorophore through charge transfer, the other part is connected with coumarin part of thiol group, hcy and Cys emit blue fluorescence through intramolecular rearrangement, and GSH cannot generate intramolecular rearrangement due to large steric hindrance, so that the GSH is combined with aldehyde group to emit green fluorescence. The fluorescence is then quenched with the C = N double bond as a reactive site for sulfite. The reaction formula is as follows:
the invention provides a method for distinguishing and detecting Cys/Hcy and GSH, which comprises the following steps:
(1) Preparing a mixture with a volume ratio of 1:1, taking a phosphate buffer solution with pH7.4 and dimethyl sulfoxide as a reaction system, dissolving MB-C in DMSO to prepare a 2mM preparation solution, and respectively preparing 20mM Glutathione (GSH), cysteine (Cys) and homocysteine (Hcy) solutions;
(2) Ultraviolet spectrum: 1960. Mu.L of the reaction system solution, 10. Mu. LMB-C and 30. Mu.L of the GSH solution were put into a cuvette and subjected to ultraviolet scanning at intervals of 90 seconds. The same procedure was performed with Cys/Hcy;
(3) Fluorescence spectrum: adding 1960 μ L of reaction system solution, 10 μ L of LMB-C, and 30 μ L of GSH solution into a cuvette, and performing fluorescence scanning at an interval of 25s; the same procedure was performed with Cys/Hcy; each mercaptan was measured three times at different excitations 380nm,445nm,630nm, respectively;
(4) Working curve: in 7 EP tubes of 2mL, 1990. Mu.L of the system solution was added; 10 mu LMB-C; then adding different amounts of GSH solution to make the concentration of GSH in the system respectively 0 μ M,10 μ M,12 μ M,14 μ M,16 μ M,18 μ M,20 μ M; shaking up and standing for 10 minutes, and performing fluorescence scanning by taking 445nm as excitation to obtain the fluorescence intensity of the sample at 529 nm; plotting and drawing by taking the concentration of the GSH as an abscissa and taking the fluorescence intensity at 529nm as an ordinate to obtain a working curve of the GSH and the MB-C; the linear regression equation is: y =114.07x +802.10 2 =0.98966; the unit of x is μ M; and performing fluorescence scanning on Cys/Hcy by taking 380nm as excitation in the same way to obtain a working curve of the fluorescence intensity of Cys/Hcy at 472nm and MB-C.
The invention provides a method for tracking GSH metabolism SO 2 A method of metabolic processes comprising the steps of:
(1) Preparing a mixture with a volume ratio of 1:1, pH7.4, and dimethyl sulfoxide as reaction system, dissolving MB-C in dimethyl sulfoxide to obtain 2mM solution, and preparing 20mM Glutathione (GSH), cysteine (Cys), homocysteine (Hcy) and sodium sulfite (Na) 2 SO 3 ) A solution; preparing a phosphate buffer solution with the pH value of 7.4 and a 2mM N-ethylmaleimide (NEM) solution;
(2) Metabolic spectroscopy: 1960 mu L of reaction system solution, 10 mu L of LMB-C and 30 mu L of GSH solution are added into a cuvette, shaken up and placed for 10 minutes; then 30. Mu.L of Na was taken 2 SO 3 Performing fluorescence scanning on the system under the conditions of 380nm,445nm and 630nm as excitation respectively for 30s; cys and Hcy were also performed;
(3) Cell application: 10 μ L of MB-C was added to 2mL of PBS solution so that the concentration thereof was 10 μ M; hela was incubated with the above solution at 37 ℃ for 10 min, and cells were washed with PBS (2 mL. Times.3) for confocal imaging; the second group treated cells with 500. Mu.M NEM for 10 min, washed with PBS and incubated with 10. Mu.M MB-C for 10 min for confocal imaging. The third group of cells was treated with 500 μ M NEM for 10 min, washed with PBS and incubated with 500 μ M Cys +10 μ MMB-C for 10 min, washed with PBS and imaged. The fourth group of cells was treated with 500. Mu.M NEM for 10 min, washed with PBS and incubated with 500. Mu.M GSH + 10. Mu.M B-C for 10 min, washed with PBS and imaged;
(4) Incubating Hela with 2mL of 500. Mu.M NEM in PBS for 10 min, washing with PBS, incubating with 500. Mu.M GSH + 10. Mu.M B-C for 10 min, washing the cells with PBS, adding 200. Mu.M Na 2 SO 3 The cell signal was monitored in real time as a function of time. No Na was added to the control group 2 SO 3 And detecting the change of the fluorescence signal of the cells. Cys operates the same as in the GSH group.
Compared with the prior art, the invention has the following beneficial effects:
1. the probe of the invention can not only distinguish and detect three kinds of biological mercaptan, but also carry out biological metabolism detection;
2. the invention has the advantages of simple technical operation and simple and convenient detection, and only needs an ultraviolet spectrophotometer and a fluorescence detector;
3. the invention has obvious detection signal, visible color change of reaction solution and rapid reaction time.
Drawings
FIG. 1 example 1 preparation of fluorescent Probe MB-C Hydrogen Spectroscopy
FIG. 2 example 1 preparation of fluorescent Probe MB-C carbon Spectroscopy
FIG. 3 Mass spectrum of fluorescent probe MB-C prepared in example 1
FIG. 4 UV spectrogram of example 2MB-C reacted with GSH
FIG. 5 UV Spectroscopy of the reaction of example 2MB-C with Cys
FIG. 6 UV spectrogram of example 2MB-C reacted with Hcy
FIG. 7,8,9 fluorescence spectra of example 3MB-C and GSH at 380nm,445nm and 630nm as excitation conditions, respectively
FIGS. 10, 11 and 12 fluorescence spectra of example 3MB-C and Cys at 380nm,445nm and 630nm as excitation conditions
FIGS. 13, 14 and 15 fluorescence spectra of example 3MB-C and Hcy under excitation conditions of 380nm,445nm and 630nm, respectively
FIG. 16 example 4 Linear relationship of MB-C to GSH fluorescence intensity at 529nm
FIG. 17 example 4 Linear relationship between fluorescence intensity of MB-C and Cys at 472nm
FIG. 18 example 4 Linear relationship between MB-C and Hcy fluorescence intensity at 472nm
FIGS. 19, 20 example 5 reaction of MB-C with GSH and Na addition 2 SO 3 Then 445nm and 630nm are respectively used as fluorescence spectrograms under the excitation condition
FIGS. 21, 22 example 5 reaction of MB-C with Cys followed by Na addition 2 SO 3 Then 380nm and 630nm are respectively used as fluorescence spectrograms under the excitation condition
FIGS. 23 and 24 addition of Na to the reaction system of example 5MB-C with Hcy 2 SO 3 Then 380nm and 630nm are respectively used as fluorescence spectrograms under the excitation condition
FIG. 25 imaging of cells incubated under different conditions in example 6MB-C
FIG. 26 example 7 reaction of MB-C with GSH with or without addition of Na 2 SO 3 The cell fluorescence intensity as a function of time
FIG. 27 example 7 whether Na was added after reaction of MB-C with Cys 2 SO 3 The fluorescence intensity of the cells of (1) is plotted against time.
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.
Example 1
Synthesis and characterization of Probe MB-C
The mixture of malonic acid (111g, 106mmol) and phenol (19.95g, 212mmol) was slowly added phosphorus oxychloride (11.5ml, 123mmol) at 0 ℃, the mixture was heated at 115 ℃ until the strong evolution of hydrogen chloride gas ceased (about 1.5 h), the upper layer was poured into 150mL of water and extracted three times with ethyl acetate, the organic phase was dried over anhydrous sodium sulfate, the solvent was dried to give diphenyl malonate (20.6 g) as a pale yellow oil.
Diphenyl malonate (12.8g, 50mmol) is dissolved in 50mL of toluene, then 3-diethylaminophenol (8.25g, 50mmol) is added, reflux reaction is carried out for 7 hours, after the reaction is finished, suction filtration is carried out, washing is carried out with petroleum ether or n-hexane, and the product is dried in vacuum to obtain light yellow solid 7- (diethylamino) -4-hydroxy-2H-chromen-2-one (8.7 g).
In an ice-water bath, N-dimethylformamide (2.8 mL) was added to phosphorus oxychloride (2.8 mL), stirred for half an hour to obtain a transparent viscous substance, and 7- (diethylamino) -4-hydroxy-2H-chromen-2-one (2.33g, 10 mmol) dissolved in N, N-dimethylformamide (13.2 mL) was added dropwise to the above solution to obtain a mauve solution. Stirring was carried out at 60 ℃ for 12 hours, then pouring into 100mL of ice water, adjusting the pH to 5 by adding sodium hydroxide to give a large amount of precipitate, suction filtration and washing with water, followed by recrystallization from anhydrous ethanol. 4-chloro-7- (diethylamino) -2-oxo-2H-chromene-3-carbaldehyde (1.2 g) was obtained.
4-chloro-7- (diethylamino) -2-oxo-2H-chromene-3-carbaldehyde (1.2g, 4.3mmol) was dissolved in 10mL of dichloromethane, stirred for 5 minutes in an ice-water bath, triethylamine (1.8mL, 12.9mmol) was added, stirring was continued for 10 minutes, p-hydroxybenzyl alcohol (0.533g, 4.3mmol) was added, the reaction was stirred for 8 hours, the solvent was removed, and the crude product was purified by silica gel column chromatography. This gave 7- (diethylamino) -4- (4- (hydroxymethyl) phenoxy) -2-oxo-2H-chromene-3-carbaldehyde (0.68 g) as a yellow product. 1 H NMR(600MHz,DMSO-d 6 )δ9.87(s,1H),7.36(d,J＝8.9Hz,1H),7.27(d,J＝7.2Hz,2H),7.06(d,J＝7.0Hz,2H),6.75(d,J＝9.1Hz,1H),6.64(s,1H),5.17(s,1H),4.44(s,2H),3.49(d,J＝6.0Hz,4H),1.13(s,6H).
7- (diethylamino) -4- (4- (hydroxymethyl) phenoxy) -2-oxo-2H-chromene-3-carbaldehyde (0.68g, 1.85mmol), 3,7-bis (dimethylamino) -10H-phenothiazine-10-carbonyl chloride (0.54g, 1.54mmol), sodium carbonate (0.59g, 5.55mmol) and 4-dimethylaminopyridine (0.19g, 1.54mmol) were dissolved in dichloromethane and the reaction was stirred under nitrogen protection in an ice bath for 8H; the solvent was removed and the crude product was purified by column chromatography on silica gel to give 4- ((7- (diethylamino) -3-formyl-2-oxo-2H-chromen-4-yl) oxy) benzyl 3,7-bis (dimethylamino) -10H-phenothiazine as a yellow solidOxazine-10-carboxylic acid salt (0.04g, 1.54mmol) was designated MB-C. 1 H NMR(600MHz,DMSO-d 6 )δ9.87(s,1H),7.37(d,J＝9.3Hz,1H),7.31(dd,J＝8.6,4.2Hz,4H),7.10(d,J＝8.6Hz,2H),6.75(d,J＝9.6Hz,1H),6.68(d,J＝2.5Hz,2H),6.66(d,J＝2.7Hz,2H),6.64(d,J＝2.6Hz,1H),5.13(s,2H),3.50–3.48(m,4H),2.88(s,12H),1.14(d,J＝6.9Hz,6H).
Example 2
Solution preparation: preparing a mixture with a volume ratio of 1:1, pH7.4 phosphate buffer solution and dimethyl sulfoxide are used as reaction system, MB-C is dissolved in dimethyl sulfoxide to prepare 2mM preparation solution, 20mM Glutathione (GSH), cysteine (Cys), homocysteine (Hcy) solution and sodium sulfite (Na) are prepared 2 SO 3 )；
Ultraviolet spectrum: 1960. Mu.L of the reaction system solution, 10. Mu. Of the LMB-C preparation solution and 30. Mu.L of the GSH solution were put into a cuvette and subjected to ultraviolet scanning at intervals of 90 seconds. Cys/Hcy was similarly manipulated to show spectral properties: with the increase of time, the absorption of the three thiols at 666nm gradually increases and then tends to be stable, but Cys and Hcy show a new absorption peak at 380nm, while the absorption peak of GSH at 445nm is weakened, and a slightly weaker absorption peak at 385nm appears. The results are shown in FIGS. 4,5 and 6.
Example 3
Solution preparation: the same as in example 2.
Fluorescence spectrum: 1960. Mu.L of the reaction system solution, 10. Mu.L of the MB-C preparation solution, and 30. Mu.L of the GSH solution were put into a cuvette and subjected to fluorescence scanning at an interval of 25 seconds. The results are shown in FIGS. 7,8 and 9. Cys/Hcy was similarly manipulated, with spectral properties such as 10, 11, 12 (13, 14, 15). Each thiol was measured three times at 380nm,445nm, and 630nm using different excitations, respectively. And (3) spectrum display: the fluorescence intensity of the three thiols at 472nm and 704nm increased with time with excitation at 380nm and 630 nm. With 445nm as excitation, cys and Hcy sharply decrease in fluorescence intensity at 498nm, while GSH sharply increases in fluorescence intensity at 529nm and then stabilizes.
Example 4
Solution preparation: the same as example 2;
working curve: in 7 EP tubes of 2mL, 1990. Mu.L of the system solution was added; 10 mu LMB-C; then adding different amounts of GSH solution to make the concentration of GSH in the system be 0 μ M,10 μ M,12 μ M,14 μ M,16 μ M,18 μ M,20 μ M. Shaking up and standing for 10 minutes, and performing fluorescence scanning by taking 445nm as excitation to obtain the fluorescence intensity of the sample at 529 nm; plotting and drawing by taking the concentration of the GSH as an abscissa and taking the fluorescence intensity at 529nm as an ordinate to obtain a working curve of the GSH and the MB-C; the linear regression equation is: y =114.07x +802.10 2 =0.98966. The unit of x is μ M; and performing fluorescence scanning by using the Cys/Hcy as excitation at 380nm to obtain the fluorescence intensity at 498nm, and processing data to obtain the working curve of the Cys/Hcy and the MB-C. The linear relationship is shown in FIGS. 16, 17, and 18.
Example 5
Metabolic spectroscopy: adding 30 μ LNa into the solution of MB-C reacted with GSH under 380nm,445nm and 630nm as excitation conditions respectively 2 SO 3 Fluorescence scans were performed at 30s intervals. Cys/Hcy was performed in the same manner. The spectral properties show that: the fluorescence intensity of the three thiols at 704nm and 472nm basically has no obvious change under the excitation of 630nm and 380nm. With 445nm as excitation, the fluorescence intensity at 529nm in the GSH system decreases sharply with time. The spectra are shown in FIGS. 19-24.
Example 6
Solution preparation: preparing phosphate buffer solution with pH of 7.4, dissolving MB-C in dimethyl sulfoxide to obtain 2mM solution, preparing 20mM Glutathione (GSH), cysteine (Cys), homocysteine (Hcy) solution, and sodium sulfite (Na) 2 SO 3 ) And 2mM N-ethylmaleimide (NEM) solution;
cell experiments: 10 μ L of MB-C was added to 2mL of PBS solution so that the concentration thereof was 10 μ M; incubating Hela with the above solution for 10 min at 37 ℃, washing the cells (2 mL × 3) with PBS for confocal imaging, the cells fluorescing in the blue, green and red channels; the second group treated cells with 500. Mu.M NEM for 10 min, washed with PBS and incubated with 10. Mu.M MB-C for 10 min, and the fluorescence of all three channels decreased. The third group of cells was treated with 500 μ M NEM for 10 minutes, washed with PBS and incubated with 500 μ M Cys +10 μ M MB-C for 10 minutes, and imaged after PBS washing, with strong fluorescence signals from both the red and blue channels, and relatively weak in the green channel. Fourth group of cells were treated with 500. Mu.M NEM for 10 min, washed with PBS and incubated with 500. Mu.M GSH + 10. Mu.M B-C for 10 min, and imaged after PBS washing, with strong fluorescence signals from all three channels. The results of all experimental groups are shown in fig. 25.
Example 7
Solution preparation: the same as in example 6;
incubation of Hela with 2mL of 500. Mu.M NEM in PBS for 10 min, washing with PBS followed by incubation with 500. Mu.M GSH + 10. Mu.M B-C for 10 min, washing of cells with PBS, addition of 200. Mu.M Na 2 SO 3 The cell signal was monitored in real time as a function of time. No Na was added to the control group 2 SO 3 And detecting the change of the fluorescence signal of the cells. Cys operates the same as in the GSH group. The results of GSH group experiments are shown in FIG. 26, incubation with Na 2 SO 3 The green fluorescence signal of the cells decreases with time. The red channel is substantially unchanged. The fluorescence signal of the control group was not substantially changed. The results of Cys experimental group and control group are shown in FIG. 27. There was no significant change in both blue and red channels for Cys experimental and control. This also confirms our spectral properties.
Claims (8)
1. A methylene blue-based near-infrared fluorescent probe MB-C is characterized in that the structural formula is as follows:
2. the method for synthesizing the methylene blue-based near-infrared fluorescent probe as claimed in claim 1, characterized by comprising the following steps:
1) Slowly adding phosphorus oxychloride into a mixture of malonic acid and phenol at 0 ℃, heating the mixture at 115 ℃ until the strong release of hydrogen chloride gas stops, pouring the upper layer into a proper amount of water, extracting with ethyl acetate for 3 times, drying an organic phase with anhydrous sodium sulfate, and spin-drying the solvent to obtain pale yellow oily diphenyl malonate; wherein the feeding molar ratio is that the molar ratio of the malonic acid to the phenol to the phosphorus oxychloride = 1.2-1.5;
2) Dissolving diphenyl malonate in toluene, adding 3-diethylaminophenol, performing reflux reaction for 7 hours, performing suction filtration, washing with petroleum ether or n-hexane, and performing vacuum drying on a product to obtain a light yellow solid 7- (diethylamino) -4-hydroxy-2H-chromene-2-one; wherein the feeding molar ratio is that the diphenyl malonate is 3-diethylaminophenol =1 to 1.2;
3) Adding N, N-dimethylformamide into phosphorus oxychloride in an ice-water bath, stirring for half an hour to obtain a transparent viscous substance, and dropwise adding 7- (diethylamino) -4-hydroxy-2H-chromene-2-one dissolved in N, N-dimethylformamide into the solution to obtain a solution; stirring for 12 hours at 60 ℃, then pouring into ice water, adding sodium hydroxide to adjust the pH value to 5 to generate a large amount of precipitate, carrying out suction filtration, washing with water, and then recrystallizing with absolute ethyl alcohol to obtain 4-chloro-7- (diethylamino) -2-oxo-2H-chromene-3-formaldehyde; wherein the feeding molar ratio of N, N-dimethylformamide to phosphorus oxychloride to 7- (diethylamino) -4-hydroxy-2H-chromen-2-one = 3-3.5;
4) Dissolving 4-chloro-7- (diethylamino) -2-oxo-2H-chromene-3-formaldehyde in dichloromethane, stirring for 5 minutes in an ice water bath, dropwise adding triethylamine, continuously stirring for 10 minutes, adding p-hydroxybenzyl alcohol, stirring for reacting for 8 hours, removing the solvent, and purifying the crude product by silica gel column chromatography to obtain a yellow product, namely 7- (diethylamino) -4- (4- (hydroxymethyl) phenoxy) -2-oxo-2H-chromene-3-formaldehyde; wherein the feeding molar ratio is 4-chloro-7- (diethylamino) -2-oxo-2H-chromene-3-formaldehyde to triethylamine to p-hydroxybenzyl alcohol = 3-3.5;
5) According to the mol ratio of 1.2-1.5:1-1.2:3-3.5:1-1.2 dissolving 7- (diethylamino) -4- (4- (hydroxymethyl) phenoxy) -2-oxo-2H-chromene-3-formaldehyde, 3,7-bis (dimethylamino) -10H-phenothiazine-10-carbonyl chloride, sodium carbonate and 4-dimethylaminopyridine in dichloromethane, and stirring for reaction for 8 hours under the conditions of nitrogen protection and ice bath; the solvent was removed and the crude product was purified by silica gel column chromatography to give 4- ((7- (diethylamino) -3-formyl-2-oxo-2H-chromen-4-yl) oxy) benzyl 3,7-bis (dimethylamino) -10H-phenothiazin-10-carboxylate as a yellow solid, named MB-C.
3. The method for synthesizing the near-infrared fluorescent probe based on the methylene blue as claimed in claim 2, wherein the molar ratio of the materials fed in the step 1) is that the materials are malonic acid, phenol, phosphorus oxychloride = 1.2; the feeding molar ratio in the step 2) is diphenyl malonate to 3-diethylaminophenol = 1.
4. The method for synthesizing the methylene blue-based near-infrared fluorescent probe according to claim 2, wherein the feeding molar ratio in the step 3) is N, N-dimethylformamide to phosphorus oxychloride: 7- (diethylamino) -4-hydroxy-2H-chromen-2-one = 3; the feeding molar ratio in the step 4) is 4-chloro-7- (diethylamino) -2-oxo-2H-chromene-3-formaldehyde to triethylamine: p-hydroxybenzyl alcohol = 3.
5. The method for synthesizing a near-infrared fluorescent probe based on methylene blue as claimed in claim 2, wherein the molar ratio of 7- (diethylamino) -4- (4- (hydroxymethyl) phenoxy) -2-oxo-2H-chromene-3-carbaldehyde, 3,7-bis (dimethylamino) -10H-phenothiazine-10-carbonyl chloride, sodium carbonate, and 4-dimethylaminopyridine in step 5) is 1.2:1:3:1.
6. the use of the near-infrared fluorescent probe of claim 1 in the preparation of a reagent for differentially detecting Cys/Hcy and GSH.
7. A method for differentially detecting Cys/Hcy and GSH using MB-C as claimed in claim 1, comprising the steps of:
(1) Solution preparation: preparing a mixture with a volume ratio of 1:1, taking a phosphate buffer solution with pH7.4 and dimethyl sulfoxide as a reaction system, dissolving the MB-C described in claim 1 in the dimethyl sulfoxide to prepare a 2mM preparation solution, and preparing a 20mM solution of Glutathione (GSH), cysteine (Cys) and homocysteine (Hcy);
(2) Ultraviolet spectrum: adding 1960 μ L of reaction system solution, 10 μ L of LMB-C, and 30 μ L of GSH solution into a cuvette, and performing ultraviolet scanning at an interval of 90s; the same procedure was performed with Cys/Hcy;
(3) Fluorescence spectrum: adding 1960 μ L of reaction system solution, 10 μ L of LMB-C, and 30 μ L of GSH solution into a cuvette, and performing fluorescence scanning at an interval of 25s; the same procedure was performed with Cys/Hcy; each thiol was measured three times with different excitations 380nm,445nm,630nm, respectively;
(4) Working curve: in 7 EP tubes of 2mL, 1990. Mu.L of the system solution was added; 10 mu LMB-C; then adding different amounts of GSH solution to make the concentration of GSH in the system respectively 0 μ M,10 μ M,12 μ M,14 μ M,16 μ M,18 μ M,20 μ M; shaking up and standing for 10 minutes, and performing fluorescence scanning by taking 445nm as excitation to obtain the fluorescence intensity of the sample at 529 nm; plotting and drawing by taking the concentration of the GSH as an abscissa and taking the fluorescence intensity at 529nm as an ordinate to obtain a working curve of the GSH and the MB-C; the linear regression equation is: y =114.07x +802.10 2 =0.98966; the unit of x is μ M; the same operation was performed for Cys/Hcy, and fluorescence scanning was performed with 380nm as excitation, to obtain a working curve of the fluorescence intensity of Cys/Hcy at 472nm with respect to MB-C.
8. Use of the near-infrared fluorescent probe as claimed in claim 1 in preparation of SO for tracking GSH metabolism 2 Use in a reagent of a pathway.
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