CN116444432B - H based on 1, 8-naphthalimide 2 S fluorescent probe and preparation method and application thereof - Google Patents

H based on 1, 8-naphthalimide 2 S fluorescent probe and preparation method and application thereof Download PDF

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CN116444432B
CN116444432B CN202310267716.7A CN202310267716A CN116444432B CN 116444432 B CN116444432 B CN 116444432B CN 202310267716 A CN202310267716 A CN 202310267716A CN 116444432 B CN116444432 B CN 116444432B
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刘文栋
朱海亮
陈鲲
章晨润
王晓敏
吕鹏程
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Hubei Nanbo Bioengineering Co ltd
Guangzhou University
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Guangzhou University
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Abstract

The invention discloses an H based on 1, 8-naphthalimide 2 S fluorescent probe and preparation method and application thereof. The H is 2 The S fluorescent probe has the following molecular structural expression, and the preparation method comprises the following steps: s1, dissolving 4-bromo-1, 8-naphthalic anhydride and 4-aminophenol in a solvent, and stirring and reacting for 12-24 hours to obtain a product 1; s2, adding the product 1, 2, 4-dinitrobenzenesulfonyl chloride and triethylamine into a solvent, and stirring for reaction for 4-12 hours to obtain a product 2; s3, adding the product 2,4- (2-aminoethyl) morpholine and a palladium catalyst into a flask, and simultaneously adding 1,1 '-binaphthyl-2, 2' -bisdiphenylphosphine and cesium carbonate for reaction to obtain H 2 S fluorescent probe. The H is 2 S fluorescent probe can rapidly and target and identify H at lower temperature 2 S gas signal molecules, and high detection sensitivity.

Description

H based on 1, 8-naphthalimide 2 S fluorescent probe and preparation method and application thereof
Technical Field
The present invention relates to a fluorescent probe, and more particularly, to a fluorescent probeH based on 1, 8-naphthalimide 2 S fluorescent probe and preparation method and application thereof.
Background
Lysosomes play an important role in various vital activities of cells, such as substance metabolism, cell membrane circulation, apoptosis. The lysosome is visualized and detected for active species, specific microenvironment and key physiological processes, so that the molecular mechanism of the lysosome participating in vital activities is well understood, and the method has important guiding significance for treatment of diseases. In recent years, many lysosome-targeting fluorescent probes, whose detection targets include hydrogen ions H, have been reported successively + Reducing species (hydrogen sulphide H 2 S, thiol compound cysteine Cys, homocysteine Hcy and glutathione GSH, etc.), active oxygen species (hydrogen peroxide H 2 O 2 Hypochlorous acid HClO, etc.), active nitrogen species (nitric oxide NO, nitrosyl hydrogen HNO, etc.), metal cations (Cu 2+ 、Zn 2+ Etc.), anions, etc. Wherein hydrogen sulfide H 2 S is an important endogenous gas signaling molecule, has an important role in regulating organelle function and stress, and is a third important gas transmitter regulating cardiovascular, neurological, immune, endocrine and gastrointestinal systems. Thus, visualise H 2 The distribution and concentration of S in biological systems will be very important and help elucidate H 2 Biological effects of S.
At present, a plurality of fluorescent probes for high-efficiency hydrogen sulfide detection have been reported by scientific researchers, and common fluorescent dyes include rhodamine, coumarin series, fluorescein, naphthalimide, boron dipyrrolidine and the like. The 1, 8-naphthalimide is a typical D-pi-A fluorescent dye, has the advantages of stable optical performance, high fluorescence quantum yield, longer fluorescence intensity, excitation wavelength and emission wavelength and the like, and is widely applied to the development of hydrogen sulfide fluorescent probes. For example, patent CN113024463A, CN114874142A, CN104610959B discloses the preparation of 1, 8-naphthalimide hydrogen sulfide fluorescent molecular probes, but in general, the fluorescent probes are combined with H 2 The reaction rate of S is affected by temperature, and the higher the temperature, the faster the reaction tends to be, but at lower in vivo temperature (37 ℃), the response rate of the fluorescent probe is affectedTo a certain limit. Therefore, not all fluorescent probes have H at in vivo temperature 2 S has the characteristics of sensitive detection, high fluorescence intensity and good biocompatibility, so that H with novel structure and simple preparation process is developed 2 S fluorescent probes are still of great research importance.
Disclosure of Invention
In order to solve the technical problems, the invention firstly provides H based on 1, 8-naphthalimide 2 S fluorescent probe. The invention introduces the group 4- (2-amino ethyl) -morpholine into H of the 1, 8-naphthalimide derivative 2 S probe, novel compound can rapidly and target and identify H at lower temperature 2 S gas signal molecules, and high detection sensitivity.
Based on the second aspect of the invention, a H based on 1, 8-naphthalimide is also provided 2 S fluorescent probe preparation method. The method has simple process, easily obtained raw materials and good industrial applicability.
Based on the third aspect of the invention, a H based on 1, 8-naphthalimide is also provided 2 S fluorescent probe for detecting H in lysosome 2 Applications of S gas signalling molecules, e.g. in endogenous hydrogen sulphide (H 2 S) application in a detection kit.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
h based on 1, 8-naphthalimide 2 The molecular structural expression of the S fluorescent probe is as follows:
h based on 1, 8-naphthalimide 2 The preparation method of the S fluorescent probe comprises the following steps:
s1, dissolving 4-bromo-1, 8-naphthalic anhydride and 4-aminophenol in a solvent, and stirring and reacting for 12-24 hours under a reflux condition; after the reaction is finished, placing the reaction liquid under ice bath, adding dilute hydrochloric acid to generate flocculent precipitate, collecting a product, cleaning and drying to obtain a product 1;
s2, mixing the product 1, 2, 4-dinitrobenzene sulfonyl chloride and triethylamine in a flask, adding a solvent, and stirring under reflux for reaction for 4-12h; purifying by column after the reaction is finished to obtain a product 2;
s3, adding the product 2,4- (2-aminoethyl) morpholine and a palladium catalyst into a flask, and simultaneously adding 1,1 '-binaphthyl-2, 2' -bisdiphenylphosphine, cesium carbonate and a solvent, and stirring and reacting under the nitrogen atmosphere, wherein the reaction condition is that the reaction is carried out for 6-24 hours at 60-100 ℃; cooling to room temperature after the reaction is finished, purifying by a column to obtain a product 3, namely H 2 S fluorescent probe.
As a preferred embodiment of the present invention, in step S1, the molar ratio of 4-bromo-1, 8-naphthalic anhydride to 4-aminophenol is 1 (1-2), e.g., 1:1.2, 1:1.4, 1:1.6, 1:1.8, etc.
As a preferred embodiment of the present invention, in step S1, the reaction temperature is 130 to 160℃such as 135℃140 ℃, 145 ℃, 150 ℃, 155℃and the like;
preferably, the reaction solvent in step S1 is 2-ethoxyethanol.
As a preferred embodiment of the invention, in step S2, the molar ratio of the products 1, 2, 4-dinitrobenzenesulfonyl chloride, triethylamine is 1 (5-10): (5-10), e.g. 1:5:10, 1:10:5, 1:6:6, 1:8:7, 1:9:8, 1:7:9, 1:6:10, 1:10:8, etc.
As a preferred embodiment of the present invention, in step S2, the reaction temperature is 30 to 80℃such as 40℃50℃60℃70℃and the like;
preferably, the reaction solvent in the step S2 is one or more of dichloromethane, chloroform and tetrahydrofuran.
As a preferred embodiment of the invention, in step S3, the molar ratio of product 2,4- (2-aminoethyl) morpholine to palladium catalyst is 1 (3-5): (0.1-0.2), e.g. 1:3:0.1, 1:4:0.1, 1:5:0.2, 1:5:0.1, 1:3:0.2, 1:4:0.2, etc.;
preferably, the palladium catalyst is selected from one or more of palladium acetate, tris (dibenzylideneacetone) dipalladium, tetrakis (triphenylphosphine) palladium.
As a preferred embodiment of the present invention, in step S3, 1 '-binaphthyl-2, 2' -bisdiphenylphosphine is added in an amount of 0.5 to 1.5 times, for example, 0.8 times, 1.0 times, 1.2 times, 1.4 times, etc., the molar amount of the palladium catalyst; the cesium carbonate is added in an amount of 10 to 15 times, for example, 11 times, 12 times, 13 times, 14 times, etc., the molar amount of the palladium catalyst.
As a preferred embodiment of the present invention, the reaction solvent in step S3 is one or more of dimethyl sulfoxide, toluene and xylene.
H based on 1, 8-naphthalimide as described hereinbefore 2 S fluorescent probe and H based on 1, 8-naphthalimide prepared by the method 2 S fluorescent probe for detecting H in lysosome 2 Use of S gas signalling molecules, in particular endogenous hydrogen sulphide (H 2 S) application in a detection kit.
H provided by the invention 2 S fluorescent probe has rich functional group properties and H 2 The maximum emission wavelength difference before and after the S response is large, the fluorescence color can be changed from green to orange, the color difference is obvious, the detection sensitivity is high, the rapid response at a lower temperature can be realized, and the method can be used for detecting endogenous H in lysosomes 2 The content and the concentration of S gas signal molecules are used for visualizing H 2 S has important role in research.
Drawings
FIG. 1 is H 2 S fluorescent probe and fluorescence intensity.
Detailed Description
The invention will now be further illustrated by means of specific examples which are given solely by way of illustration of the invention and do not limit the scope thereof.
The raw materials and reagents in the following examples of the invention were purchased from commercial products unless otherwise specified.
[ example 1 ]
(1) 70ml of 2-ethoxyethanol were added to a round bottom flask, 4-bromo-1, 8-naphthalenedicarboxylic anhydride (3.00 g,10.83 mmol) and 4-aminophenol (2.15 g,19.70 mmol) were added to the flask and stirred at 145℃under reflux overnight. The mixture was then cooled to room temperature and 50ml of 6% dilute hydrochloric acid was poured into an ice bath to form a grey precipitate, the solid was collected after filtration, washed with water and ethanol and then placed in an oven, and dried at 80 ℃ to give product 1.
1H NMR(500MHz,Chloroform-d):δ8.76(ddd,J＝25.1,15.0,3.0Hz,2H),8.43(d,J＝15.0Hz,1H),8.05(d,J＝15.0Hz,1H),7.95(t,J＝15.0Hz,1H),7.13–6.99(m,2H),6.95–6.84(m,2H),3.96(s,1H).
Product 1 (1.84 g,5.02 mmol), 2, 4-dinitrobenzenesulfonyl chloride (9.7 g,36.4 mmol) and triethylamine (3.7 g,36.6 mmol) were added to a 50mL round bottom flask. The mixture was dissolved in 20ml of dichloromethane and reacted at 40℃with stirring for 6h. The crude product obtained is purified by column purification to obtain the product 2.
1H NMR(500MHz,Chloroform-d):δ9.03(d,J＝2.9Hz,1H),8.77(dd,J＝14.9,3.0Hz,1H),8.46(ddd,J＝25.3,15.0,3.0Hz,2H),8.33(d,J＝15.0Hz,1H),8.21(d,J＝15.0Hz,1H),8.04(d,J＝15.0Hz,1H),7.85(t,J＝15.0Hz,1H),7.12–7.02(m,2H),6.92–6.83(m,2H).
Into a round bottom flask, compound 2 (1.8 g,3.01 mmol), 4- (2-aminoethyl) morpholine (1.560 g,12 mmol), palladium acetate (100.8 mg,0.45 mmol) were added to a round bottom flask containing 15mL of DMSO solvent, 1 '-binaphthyl-2, 2' -diphenylphosphine (186.6 mg,0.3 mmol) and cesium carbonate (1.75 g,5.4 mmol) were then added, the reaction was stirred under nitrogen atmosphere, reacted at 80℃for 8H, cooled to room temperature and purified by column chromatography to give Compound 3, H 2 S fluorescent probe.
1H NMR(500MHz,Chloroform-d):δ8.87(d,J＝1.9Hz,1H),8.58(dd,J＝10.3,1.9Hz,1H),8.50(dd,J＝8.1,1.2Hz,1H),8.36–8.29(m,2H),8.26(d,J＝10.5Hz,1H),7.73(t,J＝8.2Hz,1H),7.34–7.29(m,2H),7.18(d,J＝8.8Hz,1H),7.14–7.08(m,2H),5.91(t,J＝4.3Hz,1H),3.65(ddd,J＝9.5,6.0,3.3Hz,4H),3.43(td,J＝5.3,4.4Hz,2H),2.72(t,J＝5.2Hz,2H),2.51(ddd,J＝17.8,6.0,3.3Hz,4H).
[ example 2 ]
(1) 80ml of 2-ethoxyethanol was added to a round bottom flask, 4-bromo-1, 8-naphthalenedicarboxylic anhydride (3.00 g,10.83 mmol) and 4-aminophenol (1.2 g,11.0 mmol) were added to the flask and stirred at 155℃under reflux overnight. The mixture was then cooled to room temperature and 50ml of 6% dilute hydrochloric acid was poured into an ice bath to form a grey precipitate, the solid was collected after filtration, washed with water and ethanol and then placed in an oven, and dried at 80 ℃ to give product 1.
Product 1 (1.84 g,5.02 mmol), 2, 4-dinitrobenzenesulfonyl chloride (6.7 g,25.1 mmol) and triethylamine (2.6 g,25.7 mmol) were added to a 50mL round bottom flask. The mixture was dissolved in 30ml of dichloromethane and reacted at 35℃for 8 hours with stirring. The crude product obtained is purified by column purification to obtain the product 2.
Into a round bottom flask, compound 2 (1.8 g,3.01 mmol), 4- (2-aminoethyl) morpholine (1.953 g,15 mmol), palladium acetate (75.8 mg,0.34 mmol) were added to a round bottom flask containing 15mL of DMSO solvent, 1 '-binaphthyl-2, 2' -bisdiphenylphosphine (217.9 mg,0.35 mmol) and cesium carbonate (1.11 g,3.4 mmol) were then added, the reaction was stirred under nitrogen atmosphere, reacted at 60℃for 20H, cooled to room temperature and purified by column chromatography to give Compound 3, H 2 S fluorescent probe.
[ example 3 ]
(1) To a round bottom flask was added 100ml of 2-ethoxyethanol, 4-bromo-1, 8-naphthalenedicarboxylic anhydride (3.00 g,10.83 mmol) and 4-aminophenol (1.64 g,15.03 mmol) were added to the flask and stirred at 135℃under reflux overnight. The mixture was then cooled to room temperature and 50mL of 6% dilute hydrochloric acid was poured into an ice bath to form a grey precipitate, the solid was collected after filtration, washed with water and ethanol and then placed in an oven, and dried at 80 ℃ to give product 1.
Product 1 (1.84 g,5.02 mmol), 2, 4-dinitrobenzenesulfonyl chloride (13.0 g,48.8 mmol) and triethylamine (5.0 g,49.4 mmol) were added to a 50mL round bottom flask. The mixture was dissolved in 25ml of chloroform and reacted at 65℃with stirring for 12 hours. The crude product obtained is purified by column purification to obtain the product 2.
Into a round bottom flask was charged compound 2 (1.8 g,3.01 mmol), 4- (2-aminoethyl) morpholine (1.2 g,9.2 mmol), palladium acetate (134.7 mg,0.60 mmol) into a round bottom flask containing 20mL toluene solvent, then 1,1 '-binaphthyl-2, 2' -bisdiphenylphosphine (560.4 mg,0.9 mmol) and cesium carbonate (2.935 g,9 mmol) were added under nitrogenStirring and reacting in air atmosphere, reacting at 100deg.C for 12 hr, cooling to room temperature, purifying with column to obtain compound 3, namely H 2 S fluorescent probe.
[ application example ]
(1) H of the probe 2 S response test:
first 1mmol/L of H prepared in example 1 was prepared 2 S fluorescent probe aqueous solution 1. Mu. Mol/LNaHS solution (as H 2 S donor) was added to the probe solution and the maximum absorption wavelength, maximum emission wavelength, stokes shift before and after NaHS addition were detected, and the test results are shown in table 1:
TABLE 1, H 2 S response test results
Before NaHS is added After NaHS addition
Maximum absorption wavelength 508nm 522nm
Maximum emission wavelength 552nm 634nm
Stokes shift 44nm 112nm
As can be seen from the above test results, the direction isH prepared in example 1 2 NaHS solution is added into the S fluorescent probe aqueous solution, the maximum absorption wavelength, the maximum emission wavelength and Stokes shift of the compound are all obviously changed, and especially the maximum emission wavelength is changed from 552nm (green) to 634nm (orange), which indicates that the fluorescent detection accuracy is high.
It is also notable that H prepared in example 1 2 The background fluorescence of the aqueous solution of S fluorescent probe was very weak (Φ=0.03), and a high fluorescence intensity (Φ=0.32) was observed within 5 minutes after addition of NaHS solution, indicating that the fluorescent probe was compatible with H 2 S reacts to increase the fluorescence intensity; thus, the fluorescent probe can detect H in real time in intracellular imaging 2 S gas signal molecules.
(2) Fluorescence intensity test at specific temperature:
to 1mmol/L H of the formulation 2 The solution shows gradually enhanced fluorescence response within 30min under the irradiation of 365nm ultraviolet light by adding 1 mu mol/L NaHS solution into the aqueous solution of the S fluorescent probe, and particularly, high fluorescence intensity is detected within 5min, which indicates H 2 The S detection sensitivity is high. The continuous detection results are shown in table 2:
TABLE 2 fluorescent intensity test results at specific temperatures
Time/min 0 5 10 15 20 25 30 35 40
Fluorescence intensity/a.u. 4 67 130 179 235 277 279 280 281
As can be seen from the test results in Table 2, the fluorescent probe provided by the invention can complete H rapidly even at in vivo temperature 2 S response (complete response within 25 min), indicating that it is significantly more suitable for live cell fluorescence imaging and endogenous H in this regard 2 Application of S gas signal molecule targeted fluorescent probe.
(3) Relationship between substance concentration and fluorescence intensity
Preparation of H at different concentrations according to the data in Table 3 2 S fluorescent probe aqueous solution, and 1. Mu. Mol/L NaHS solution was added respectively, and fluorescence intensity was measured under the irradiation of 365nm ultraviolet light at 37℃and standard intensity (F/F) was recorded 0 ) Wherein F 0 The fluorescence intensity before NaHS addition and F the fluorescence intensity 25min after NaHS addition. The results are shown in Table 3:
TABLE 3 relationship between substance concentration and fluorescence intensity
Concentration (mu mol/L) 5 10 20 30 40 50 60 80 100
Standard intensity (F/F) 0 ) 0.8 1.8 3.2 4.1 4.9 5.8 7.1 7.9 9.2
The test data in Table 3 is plotted as a function, as shown in FIG. 1, and it can be seen thatH in a certain concentration 2 S fluorescent probe aqueous solution, fluorescence intensity of the solution and H 2 The concentration of the S fluorescent probe is in a linear positive correlation.
(4) Detection sensitivity test:
the fluorescent probe solutions (final concentration: 1. Mu. Mol/L) were added to NaHS solutions of different concentrations (final concentrations: 5, 10, 25, 50, 100, 250 nmol/L) to carry out a reaction, and the fluorescence spectra of the solutions after the reaction were measured. The result shows that the fluorescent probes generate the change of the fluorescent signal of a turn on type when reacting with NaHS with different concentrations (in the range of 5-250 nmol/L), which indicates that the fluorescent detection sensitivity is high and the application range is wide.
(5) Fluorescence selectivity test:
adding the following anionic compounds which are common in nature or human body, including KCl, naI, mgSO, into 1mmol/L fluorescent probe aqueous solution 4 、KNO 3 、NaNO 2 、Na2CO 3 、NaHCO 3 NaOAc and commonly active small molecules, including glutathione, cysteine, glutamic acid, proline, serine, 5-aminolevulinic acid, arginase (1. Mu. Mol/L), found that the fluorescence emission intensity of these solution samples was not significantly changed compared to that of the fluorescence probe alone (phi=0.03), indicating that the fluorescence probe was specific for H only 2 S has a high degree of responsiveness and selectivity.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and additions may be made to those skilled in the art without departing from the method of the present invention, which modifications and additions are also to be considered as within the scope of the present invention.

Claims (12)

1. H based on 1, 8-naphthalimide 2 An S fluorescent probe, characterized in that the H 2 The molecular structural expression of the S fluorescent probe is as follows:
2. h based on 1, 8-naphthalimide 2 The preparation method of the S fluorescent probe is characterized by comprising the following steps:
s1, dissolving 4-bromo-1, 8-naphthalic anhydride and 4-aminophenol in a solvent, and stirring and reacting for 12-24 hours under a reflux condition; after the reaction is finished, placing the reaction liquid under ice bath, adding dilute hydrochloric acid to generate flocculent precipitate, collecting a product, cleaning and drying to obtain a product 1;
s2, mixing the product 1, 2, 4-dinitrobenzene sulfonyl chloride and triethylamine in a flask, adding a solvent, and stirring under reflux for reaction for 4-12h; purifying by column after the reaction is finished to obtain a product 2;
s3, adding the product 2,4- (2-aminoethyl) morpholine and a palladium catalyst into a flask, and simultaneously adding 1,1 '-binaphthyl-2, 2' -bisdiphenylphosphine, cesium carbonate and a solvent, and stirring and reacting under the nitrogen atmosphere, wherein the reaction condition is that the reaction is carried out for 6-24 hours at 60-100 ℃; cooling to room temperature after the reaction is finished, purifying by a column to obtain a product 3, namely H 2 S fluorescent probe.
3. 1, 8-naphthalimide-based H according to claim 2 2 A method for producing an S fluorescent probe, characterized in that in step S1, the molar ratio of 4-bromo-1, 8-naphthalic anhydride to 4-aminophenol is 1 (1-2).
4. A 1, 8-naphthalimide-based H according to claim 3 2 The preparation method of the S fluorescent probe is characterized in that in the step S1, the reaction temperature is 130-160 ℃.
5. The 1, 8-naphthalimide-based H of claim 4 2 The preparation method of the S fluorescent probe is characterized in that the reaction solvent in the step S1 is 2-ethoxyethanol.
6. 1, 8-naphthalimide-based H according to any of claims 2-5 2 The preparation method of the S fluorescent probe is characterized in that in the step S2, the molar ratio of the product 1 to the 2, 4-dinitrobenzenesulfonyl chloride to the triethylamine is 1 (5-10): 5-10.
7. The 1, 8-naphthalimide-based H of claim 6 2 The preparation method of the S fluorescent probe is characterized in that in the step S2, the reaction temperature is 30-80 ℃.
8. 1, 8-naphthalimide-based H of claim 7 2 The preparation method of the S fluorescent probe is characterized in that the reaction solvent in the step S2 is one or more of dichloromethane, chloroform and tetrahydrofuran.
9. 1, 8-naphthalimide-based H according to any of claims 2-5 2 The preparation method of the S fluorescent probe is characterized in that in the step S3, the molar ratio of the product 2 to the 4- (2-amino ethyl) morpholine to the palladium catalyst is 1 (3-5) (0.1-0.2).
10. 1, 8-naphthalimide-based H according to claim 9 2 The preparation method of the S fluorescent probe is characterized in that the palladium catalyst is selected from one or more of palladium acetate, tris (dibenzylideneacetone) dipalladium and tetrakis (triphenylphosphine) palladium.
11. 1, 8-naphthalimide-based H according to claim 9 2 The preparation method of the S fluorescent probe is characterized in that in the step S3, the addition amount of 1,1 '-binaphthyl-2, 2' -bisdiphenylphosphine is 0.5 to 1.5 times of the molar amount of the palladium catalyst; the adding amount of cesium carbonate is 10-15 times of the molar amount of palladium catalyst.
12. 1, 8-naphthalimide-based H of claim 11 2 The preparation method of the S fluorescent probe is characterized in that the reaction solvent in the step S3 is dimethylOne or more of sulfoxide, toluene and xylene.
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CN113024463A (en) * 2021-03-07 2021-06-25 天津大学 Preparation and application of 1, 8-naphthalimide hydrogen sulfide fluorescent molecular probe
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