CN114591264A

CN114591264A - Fluorescent probe for indicating pH value and preparation method and application thereof

Info

Publication number: CN114591264A
Application number: CN202011404399.1A
Authority: CN
Inventors: 不公告发明人
Original assignee: Hunan Chaoji Testing Technology Co ltd
Current assignee: Hunan Chaoji Testing Technology Co ltd
Priority date: 2020-12-04
Filing date: 2020-12-04
Publication date: 2022-06-07

Abstract

The invention provides a fluorescent probe capable of indicating a pH value through color, and a preparation method and application thereof. The chemical structural formula of the fluorescent probe is as follows:

Description

Fluorescent probe for indicating pH value and preparation method and application thereof

Technical Field

The invention belongs to the technical field of analytical chemistry, and relates to a fluorescent probe for indicating a pH value, and a preparation method and application thereof.

Background

The change of pH value is closely related to the progress of many biological reactions, and the nucleic acid detection technology, which is an important technology in molecular biology, is also closely related to the change of pH value, wherein loop-mediated isothermal amplification (LAMP) is favored by researchers due to its higher sensitivity and faster reaction rate. Loop-mediated isothermal amplification (LAMP) involves a large amount of DNA replication during the reaction, consumes a large amount of deoxyribonucleotide triphosphates (dNTPs), and generates a large amount of pyrophosphate ions when the dNTPs are consumed, which causes a decrease in pH of the entire reaction system. Therefore, whether or not the amplification reaction is carried out containing the target nucleic acid can be determined by observing the change in pH.

A common pH value detection method is a glass electrode method, but the method has the defects of electrochemical interference, large size and the like, and the pH test paper detection method is only suitable for rough measurement, is not suitable for trace detection and can not achieve the aim of nucleic acid detection; the method for detecting intracellular pH comprises³¹P nuclear magnetic resonance method, absorption spectroscopy, etc., but these methods have disadvantages such as mechanical damage and high price. Although the above conventional methods can be used for pH detection, these methods usually require sample pretreatment and cell destruction, are complicated to operate, and have limitations in biological studies. The pH detection method based on fluorescence has quick response, and can realize the change from red light to yellow and green light in the process of changing the pH value from 4 to 9, thereby realizing the detection of the pH value.

Disclosure of Invention

The present invention is directed to overcoming the above-mentioned drawbacks of the prior art and providing a fluorescent probe for detecting pH, which can detect pH in the range of 4 to 9 by changing fluorescence.

In order to achieve the purpose, the invention adopts the following technical scheme.

A fluorescent probe for indicating pH, the fluorescent probe having a chemical formula 1:

formula 1 chemical structural formula

The preparation method of the fluorescent probe for indicating the pH value comprises the following steps:

(1) adding 2- (N-morpholino) ethanesulfonic acid (compound 1) into anhydrous dichloromethane solution/chloroform/dichloroethane, dropwise adding oxalyl chloride and dimethylformamide, placing into an ice water bath under the protection of nitrogen, fully stirring, after complete reaction, filtering to remove filter residue, and concentrating the filtrate to obtain compound 2.

(2) Taking isatoic anhydride, glacial acetic acid and 2-aminothiophenol to react in a water bath environment at the temperature of 45 ℃. After the reaction is finished, cooling to room temperature, pouring into pure water to separate out a tawny precipitate, and adjusting the pH value of the system to be nearly neutral by using a sodium hydroxide solution to separate out a large amount of tawny precipitate. Filtration under reduced pressure gave a solid precipitate which was washed 3 times with saturated sodium chloride solution. The collected filter cake was dried in an oven at 50 ℃. Then column chromatography separation and purification are carried out to obtain a yellow brown product, namely the compound 3.

(3) Dissolving the compound 3 in anhydrous dichloromethane/trichloromethane/chloroform/dichloroethane, and adding a certain amount of triethylamine solution. The mixture was cooled to 0 ℃, compound 2 was added dropwise in anhydrous dichloromethane, and the reaction was stirred to completion at room temperature. And (3) carrying out pure water quenching reaction, collecting an organic layer, drying, carrying out reduced pressure rotary evaporation to remove the solvent, and carrying out column chromatography purification to obtain the compound 4.

(4) Dissolving the compound 4 in acetic anhydride, slowly adding nitric acid and glacial acetic acid dropwise at 50 ℃, and stirring for reaction. After the reaction was complete, the excess acid was neutralized using NaOH solution. The resulting mixture was extracted with dichloromethane, the organic layer was dried over anhydrous magnesium sulfate, and the residual solvent was removed under reduced pressure to give a crude nitro compound as a pale yellow solid. Finally the crude product was dissolved in tetrahydrofuran and 10% Pd/C powder was added and stirred at room temperature for a while. After filtration, the solvent was removed under reduced pressure to give final product ABT-Lys.

Another object of the present invention is to provide the use of the above fluorescent probe for pH value indication in nucleic acid detection.

The mechanism of the invention is as follows:

the probe provided by the invention is provided with a 2- (2' -aminophenyl) benzothiazole group, when the pH value gradually changes to an acidic condition, the amino hydrogen on the probe and the nitrogen atom of the benzothiazole form an intramolecular hydrogen bond to form an ESIPT process, and at the moment, red fluorescence is enhanced; in the alkaline environment, the ESIPT process is blocked to emit yellow-green light; the detection of the pH value can be achieved by detecting the color change at a certain wavelength using a fluorescence detector.

The invention has the following beneficial effects:

compared with the conventional trace pH value detection method, the method can distinguish different colors by naked eyes, and realize the detection of the pH value; the synthetic method is simple, the steps are simple, and the operation is simple and convenient; has wide application prospect.

Drawings

FIG. 1: preparation of Compound 4 in example 1¹H NMR spectrum;

FIG. 2: preparation of Compound 4 in example 1¹³A C NMR spectrum;

FIG. 3: the HR-MS profile of compound 4 of example 1;

FIG. 4: method for preparing ABT-Lys fluorescent Probe in example 1¹H NMR spectrum;

FIG. 5: method for preparing ABT-Lys fluorescent Probe in example 1¹³A C NMR spectrum;

FIG. 6: HR-MS profile of ABT-Lys fluorescent probe of example 1;

FIG. 7: fluorescence emission spectra of the pH-indicating fluorescent probes of example 2 at pH 4-9, respectively;

FIG. 8: the fluorescent probes indicating pH in example 2 exhibited different colors at pH 4 to 9, respectively;

FIG. 9: the application of the fluorescent probe in the 2019-nCoV visual rapid detection kit in example 3 is shown in the figure.

Detailed Description

Example 1 Synthesis of fluorescent probes indicating pH

(1) Synthesis of Compound 2: to a solution of 2- (N-morpholino) ethanesulfonic acid (2.1 g, 10 mmol) in anhydrous dichloromethane (50.0 mL) under nitrogen, oxalyl chloride (1.7 mL, 20.0 mmol) and three drops of anhydrous dimethylformamide were added dropwise. The reaction mixture was stirred for 0.5 hours under an ice-water bath, then allowed to reach room temperature and stirred for 5 hours. After the reaction, the mixture was filtered to remove insoluble solids. The solvent was then removed under vacuum to give 1.4 g of compound 2 by mass, in 67.3% yield.

(2) Synthesis of Compound 3: 1.0 g of isatoic anhydride was weighed, placed in a 50 mL round-bottom flask, and added with 15 mL of glacial acetic acid, and stirred at room temperature. 0.85 mL of 2-aminothiophenol is weighed and added into the reaction system, then the reaction system is moved to the water bath environment, the temperature of the water bath is set to be 45 ℃, and the reaction time is 2 hours. As the reaction proceeded, the reaction system gradually changed from a turbid state to a clear state, and the color gradually changed from grayish brown to orange yellow. TLC monitors the reaction process, and the dark spots of the raw material are gradually reduced to generate blue fluorescent spots with lower polarity and fewer byproducts. After the reaction is finished, the reaction system is moved to the room temperature environment for cooling, and is poured into 200 mL of purified water to separate out a tawny precipitate, and a large amount of tawny precipitate is separated out after the pH of the system is adjusted to be nearly neutral by using 1 mol/L sodium hydroxide solution. The solid precipitate was obtained by filtration under reduced pressure and washed 3 times with 100 mL of saturated sodium chloride solution, and the collected filter cake was placed in an oven and dried to remove water at 50 ℃. Then, the completely dried filter cake is taken out, and column chromatography separation and purification are carried out by adopting silica gel to obtain 1.1 g of a tawny product with the yield of 80.1 percent,¹H NMR (500 MHz, CDCl₃) δ 8.01 (d, J =8.1 Hz, 1H), 7.90 (d, J = 7.9 Hz, 1H), 7.75 (d, J = 7.9 Hz, 1H), 7.49 (t, J = 7.6 Hz, 1H), 7.39 (t, J = 7.5 Hz, 1H), 7.26 (t, J = 7.6 Hz, 1H), 6.82 (d, J = 8.2 Hz, 1H), 6.78 (t, J = 7.5 Hz, 1H), 6.42 (s, 2H). ¹³C NMR (125 MHz, CDCl₃) δ C169.27, 153.77, 146.78, 133.31, 131.59, 130.36, 126.05, 124.88, 122.46, 121.21, 116.93, 116.83, 115.32, compound 3.

(3) Synthesis of Compound 4: compound 3 (1.1 g, 5 mmol) was dissolved in anhydrous dichloromethane (40.0 mL), and triethylamine (1.4 mL, 10 m) was addedmol). The mixture was cooled to 0 ℃, and then a solution of prepared compound 2 in anhydrous dichloromethane (20.0 mL) was added dropwise, then allowed to reach room temperature and stirred for 3 hours. The reaction mixture was quenched with water (200 mL), and the organic layer was collected and dried over anhydrous magnesium sulfate. After removal of the solvent by evaporation, the residue was purified by column chromatography (silica gel, hexane: ethyl acetate 1: 1, v/v) to give a white solid (1.05 g, 52.6%),¹H NMR (400 MHz, DMSO) δ 11.80 (s, 1H), 8.22 (d, J = 8.0 Hz, 1H), 8.05 (d, J = 8.0 Hz, 2H), 7.74 (d, J = 8.3 Hz, 1H), 7.60 (d, J = 8.3, 1.3 Hz, 2H), 7.57 – 7.52 (m, 1H), 7.34 – 7.28 (m, 1H), 3.54 (t, J = 6.6 Hz, 2H), 3.17 (s, 4H), 2.65 (t, J= 6.6 Hz, 2H), 2.11 (s, 4H). ¹³C NMR (101 MHz, DMSO) δ 168.37, 152.34, 137.51, 133.32, 132.94, 130.99, 127.65, 126.72, 124.10, 122.82, 122.80, 119.70, 118.78, 65.99, 53.29, 52.67, 48.40. HR-MS (m/z): calculated for C₂₄H₁₇N₃O₄S₂[M+H]⁺404.1103, found, 404.1155, Compound 4; it is provided with¹The H NMR spectrum is shown in a figure 1,¹³the C NMR spectrum is shown in figure 2, and the HR-MS spectrum is shown in figure 3.

(4) Synthesis of ABT-Lys: compound 4 (806 mg, 2 mmol) was dissolved in 20 mL of acetic anhydride and stirred at 50 ℃. 0.6 mL of nitric acid and 2.0 mL of glacial acetic acid were added dropwise to the above solution. After the reaction was completed, the excess acid was neutralized using NaOH solution (5 mol/L). The mixture was extracted with dichloromethane, and the organic layer was dried over anhydrous magnesium sulfate. The solvent was removed under reduced pressure to give 547 mg of crude nitro compound as a pale yellow solid. The crude product was dissolved in tetrahydrofuran (100 mL) and 10% Pd/C powder (10 mg) was added. Then, the resulting solution was stirred at room temperature under hydrogen for 5 hours. After filtration, the solvent was removed under reduced pressure. The residue was purified by column chromatography (silica gel, hexane: ethyl acetate 1: 1, v/v) to give a yellow solid (233 mg, 27.9%)¹H NMR (500 MHz, DMSO-d₆) δ 10.45 (s, 1H), 8.18 (d, J = 7.9 Hz, 1H), 8.04 (d, J = 7.9 Hz, 1H), 7.59 – 7.55 (t, J = 7.2 Hz, 1H), 7.52 – 7.48 (t, J = 7.2, 1H), 7.35 (d, J = 8.7 Hz, 1H), 7.31 (d, J = 2.6 Hz, 1H), 6.79 (dd, J = 8.7, 2.6 Hz, 1H), 5.49 (s, 2H), 3.35 – 3.30 (m, 4H), 3.27 – 3.23 (m, 2H), 2.55 – 2.51 (m, 2H), 2.10 (s, 4H).¹³C NMR (125 MHz, DMSO-d₆) δ 167.75, 152.47, 147.20, 134.29, 127.27, 126.28, 125.34, 124.99, 124.78, 122.87, 122.64, 118.05, 114.30, 66.22, 53.19, 52.25, 48.21. HR-MS (m/z): calculated for C₂₄H₁₇N₃O₄S₂[M+H]⁺419.1212, found, 419.1190, it¹The H NMR spectrum is shown in figure 4,¹³the C NMR spectrum is shown in FIG. 5, and the HR-MS spectrum is shown in FIG. 6.

Example 2 response of the prepared fluorescent probes to pH

7 probe buffer solutions with a concentration of 20. mu.M were prepared in advance, and then the probe buffer solutions were added to PBS aqueous solutions with pH values of 4, 5, 6, 7, 7.4, 8, and 9, respectively, before fluorescence detection was performed. When the fluorescent probe is irradiated at lambda ex = 365 nm, the maximum emission wavelength is blue-shifted from 621 nm to 555 nm, the color change can be seen by naked eyes, as the pH value becomes lower, the yellow-green light is shifted to the red light, the probe has good response to aqueous solutions with different pH values, and therefore, the fluorescent probe can be used for indicating the pH value; the fluorescence spectra at different wavelengths are shown in fig. 7, and the different color changes are shown in fig. 8 when irradiated at λ ex = 365 nm.

Application of fluorescent probe prepared in example 3 in 2019-nCoV visual rapid detection kit

And transferring 2.5 muL +10 muL enzyme preparation +5 muL sterile water +2.5 muL primer into a 2019-nCoV rapid detection kit with a concentration of 20 muM, wherein the total amount is 6. Positive control: adding 5 muL of pseudovirus with the concentration of 5000 copy/mL (three parts in total); negative control substance: 5 muL of sterile water (three parts in total) is added into the sample, and the sample is placed in a constant-temperature water bath at 65 ℃ and heated for 45 min and then taken out. The probe can be used in a 2019-nCoV visual rapid detection kit for application as shown in FIG. 9 because the color of a positive control added with the pseudovirus is changed from yellow green to red and a negative control added with sterile water is still yellow green when the probe is observed by irradiation at 365 nm.

Claims

1. A fluorescent probe capable of indicating pH value by color has a chemical structural formula shown in formula 1:

the chemical structural formula of the formula 1.

2. A method for preparing a fluorescent probe according to claim 1, characterized by the following steps:

(1) adding 2- (N-morpholino) ethanesulfonic acid into anhydrous dichloromethane solution/chloroform/dichloroethane, dropwise adding oxalyl chloride and dimethylformamide, stirring in ice water bath under the protection of nitrogen gas, filtering to remove filter residue and concentrating the filtrate to obtain compound 2;

(2) dissolving the compound 3 in anhydrous dichloromethane/trichloromethane/chloroform/dichloroethane, and adding a certain amount of triethylamine solution; cooling the mixture to 0 ℃, dropwise adding the compound 2 into anhydrous dichloromethane, and stirring at room temperature to react completely; carrying out pure water quenching reaction, collecting an organic layer, drying, carrying out reduced pressure rotary evaporation to remove a solvent, and carrying out column chromatography purification to obtain a compound 4;

(3) dissolving a compound 4 in acetic anhydride, slowly dropwise adding nitric acid and glacial acetic acid at 50 ℃, and stirring for reaction; after the reaction is finished, neutralizing excessive acid by using NaOH solution; extracting the obtained mixture with dichloromethane, and drying the organic layer with anhydrous magnesium sulfate; removing the residual solvent under reduced pressure to give a crude nitro compound as a pale yellow solid; finally, the crude product was dissolved in tetrahydrofuran, and the synthetic route was as follows:

3. use of a fluorescent probe according to claims 1-2, characterized in that: can be used for detecting pH value in solution, nucleic acid or organism.