CN110295041B

CN110295041B - SO (SO)2Ratio type fluorescent probe and preparation method and application thereof

Info

Publication number: CN110295041B
Application number: CN201910724061.5A
Authority: CN
Inventors: 林伟英; 张天歌; 朱琳琳
Original assignee: University of Jinan
Current assignee: University of Jinan
Priority date: 2019-08-07
Filing date: 2019-08-07
Publication date: 2020-07-17
Anticipated expiration: 2039-08-07
Also published as: CN110295041A

Abstract

The invention provides a fluorescent probe for detecting sulfur dioxideNeedle:

. The probe can specifically detect bisulfite in solution and cells, and has a deep red emission spectrum and strong anti-interference performance.

Description

SO (SO)2Ratio type fluorescent probe and preparation method and application thereof

Technical Field

The invention belongs to the technical field of analytical chemistry, and particularly relates to a fluorescent probe for detecting sulfur dioxide and application thereof.

Background

SO₂Is one of the indispensable active sulfur existing in the living body, and plays an important role in various physiological and pathological processes in human. Endogenous SO₂Is mainly due to H in the mitochondria of cells₂S and sulfur-containing small molecules (such as cysteine (Cys) and homocysteine (Hcy)) undergo catalytic oxidation reaction under the action of enzyme. SO (SO)₂Is an important factor for regulating cardiovascular diseases, has the functions of relaxing blood vessels, reducing blood pressure, resisting oxidation and the like, and abnormal values of the factor can cause a plurality of health disorders, for example, excessive SO intake₂Can cause respiratory diseases (including asthma, emphysema and chronic bronchitis), cranial nerve disorders (including migraine, apoplexy and brain cancer) and even lung cancer. Thus, real-time monitoring of SO in living cells₂The level of (A) has a very important meaning for the diagnosis of the relevant diseases.

For conventional SO₂Detection methods such as high performance liquid chromatography, electrochemical detection, mass spectrometry, colorimetry and capillary electrophoresis have been reported, but they also have many disadvantages such as high cost, complexity, difficulty in understanding, tissue or cell destruction, and complicated operation, thus limiting the practical application of these methods. In contrast, the fluorescence bioimaging method has significant advantages, and has the advantages of simple operation, high selectivity and sensitivity, low detection limit, real-time, nondestructive, intracellular detection and the like. Thus, the design identifies the SO₂The fluorescent probe has important significance. The ratio type fluorescent probe has higher accuracy and sensitivity than the 'enhancement type' and 'quenching type' fluorescent probes because the false positive result caused by environmental interference is eliminated.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides the fluorescent probe for detecting sulfur dioxide, which has the advantages of high response speed and strong anti-interference capability.

Another object of the present invention is to provide an application of the above fluorescent probe in detecting bisulfite in solution and in biological cells.

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

A fluorescent probe for detecting sulfur dioxide has a chemical structural formula shown in formula (I):

formula (I).

The preparation method of the fluorescent probe comprises the following steps:

（1）N₂under the protection of gas, under the condition of existence of NaOH and KI, heating 2-methoxyphenothiazine and 1-bromobutane in DMSO for reflux reaction, and purifying reaction liquid to obtain a light yellow oily compound 1;

(2) at 0 ℃ N₂Under the protection of gas, when phosphorus oxychloride and DMF are stirred until light pink solid appears, adding a DMF solution of a compound 1, heating for reaction, and purifying a reaction solution to obtain a yellow solid compound 2;

(3) dichloromethane solution of compound 2 and AlCl at 0-5 deg.C₃Mixing, reacting at room temperature, and purifying the reaction solution to obtain a yellow oily compound 3;

（4）N₂under the protection of gas, the compound 3 and the p-aminobenzonitrile react in ethanol at room temperature, and the reaction liquid is purified to obtain orange red solid, namely the fluorescent probe.

In the step (1), the reaction temperature is 95 ℃.

In the step (2), the reaction temperature is 60 ℃.

An application of the fluorescent probe in detecting bisulfite in solution, cells or organisms.

The mechanism of the invention is as follows:

the fluorescence probe phenothiazine fluorophore has large Stokes shift and long emission wavelength, N atoms and S atoms both have lone pair electrons and are introduced into the structure of a target molecule as stronger electron donating groups, and CN groups are used as electron withdrawing groups to form a large electron push-pull system with phenothiazine groups, so that the probe Ph-CN with ICT effect is obtained. In the presence of bisulfite radicals, C = N on the probe generates Michael addition reaction, cuts off a large conjugated structure of a system, blocks ICT effect and generates Ph-CN-SO₂The deep red emission at 660 nm attenuates the blue fluorescence enhancement at 460 nm.

The invention has the following advantages:

the deep red emission probe Ph-CN can effectively avoid self-absorption and autofluorescence of biological tissues, and has strong biological tissue penetrability. The probe Ph-CN has good selectivity and stability, and is used for treating SO₂Has a fast response speed and is resistant to SO in the acidic and neutral ranges₂Good response, can generate larger emission displacement and avoid the emission displacement with SO₂The spectra of the products after the interaction are overlapped, which is beneficial to the further application of the probe in a living biological system.

Drawings

FIG. 1 shows a fluorescent probe¹H NMR spectrum;

FIG. 2 shows a fluorescent probe¹³A C NMR spectrum;

FIG. 3 is a mass spectrum of a fluorescent probe;

FIG. 4 is the response of a fluorescent probe to different concentrations of bisulfite;

FIG. 5 is the kinetics of the response of a fluorescent probe to bisulfite;

FIG. 6 is the selectivity of fluorescent probes for different ions, small molecules and amino acids;

FIG. 7 is a graph of the effect of different pH on probes and probe response to bisulfite;

FIG. 8 is an imaging application of fluorescent probes in living cells.

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 of fluorescent Probe

(1) Synthesis of Compound 1:

2-methoxyphenothiazine (1.145 g, 5 mmol), 1-bromobutane (1.36 g, 10 mmol), potassium iodide (12 mg, 0.072 mmol) and sodium hydroxide (0.4 g, 10 mmol) were weighed into a round-bottomed flask, dissolved in an appropriate amount of DMSO, and N was added₂Stirring for 6 hours at 95 ℃ under the protection of gas, stopping the reaction, cooling to room temperature, pouring the product into water, extracting with dichloromethane, washing with sodium chloride and water for three times respectively, collecting an organic phase, drying with anhydrous sodium sulfate, removing the solvent by reduced pressure distillation, and purifying by column chromatography (petroleum ether: dichloromethane = 30: 1 v/v) to obtain a pale yellow oily product with a yield of 71%;

(2) synthesis of Compound 2:

at 0 ℃ N₂Under the protection condition, dropwise adding dry DMF (464 mu L, 6 mmol) into phosphorus oxychloride (559 mu L, 6 mmol) until light pink solid appears, adding a proper amount of DMF solvent-dissolved compound 1 (1.145 g, 5 mmol), heating to 60 ℃, stirring for 4 hours, stopping reaction, cooling to room temperature, pouring the product into 100 g of crushed ice, adjusting the pH to 7 with sodium bicarbonate, extracting with dichloromethane, washing with sodium chloride and water for three times respectively, collecting an organic phase, drying with anhydrous sodium sulfate, distilling under reduced pressure to remove the solvent, purifying by column chromatography (petroleum ether: ethyl acetate = 15: 1 v/v) to obtain yellow solid with the yield of 65%;

(3) synthesis of Compound 3:

weighing compound 2 (626 mg, 2 mmol) and dissolving in appropriate amount of dry dichloromethane solution, adding aluminum trichloride (1.064 g, 8 mmol), N₂Stirring for 15 minutes at 0-5 ℃ under the protection of gas, then reacting for 6 hours at room temperature, after the reaction is finished, pouring the product into 50 m L ice water, extracting with dichloromethane, washing sodium chloride and water respectively for three times, collecting an organic phase, drying with anhydrous sodium sulfate, removing a solvent by reduced pressure distillation, and purifying by column chromatography (petroleum ether: dichloromethane = 5: 1 v/v) to obtain a yellow oily product with the yield of 61%;

(4) synthesis of Probe Ph-CN:

weighing compound 3 (299 mg, 1 mmol), dissolving in appropriate amount of dry ethanol solution, adding p-aminobenzonitrile (92.5 mg, 1.2 mmol), N₂Stirring for 5 hours at room temperature under the protection of air, stopping the reaction, filtering the product, and washing the product with ethanol to obtain an orange-red solid with the yield of 70 percent;

it is composed of¹The H NMR spectrum is shown in figure 1:¹H NMR (400 MHz, DMSO-d ₆) 12.94 (s, 1H), 8.81 (s,1H), 7.89 (d,J= 8.5 Hz, 2H), 7.50 (d,J= 8.5 Hz, 2H), 7.38 (s, 1H), 7.24 –7.15 (m, 2H), 7.08 (d,J= 8.1 Hz, 1H), 6.99 (t,J= 7.4 Hz, 1H), 6.57 (s,1H), 3.92 (t,J= 6.9 Hz, 2H), 1.73 – 1.64 (m, 2H, 1.39 (dt,J= 14.6, 7.4Hz, 2H), 0.89 (t,J= 7.4 Hz, 3H)）；

it is composed of¹³The C NMR spectrum is shown in FIG. 2:¹³C NMR (100MHz, DMSO-d ₆) 163.75, 162.50 , 152.72,150.65, 143.19 , 134.14, 130.36, 128.13, 127.57, 123.70, 122.68, 117.02,114.65, 113.23, 108.74, 103.89, 47.20, 28.66, 19.78, 14.06；

its HRMS spectrum is shown in figure 3: HRMS (M/z) [ M + H ]]⁺calcd for C₂₄H₂₂N₃OS⁺, 400.1405;found, 400.1478。

Example 2 response of fluorescent probes to different concentrations of bisulfite

The detection SO of the invention is prepared at a concentration of 1 mM₂The ratio-based fluorescent probe Ph-CN in DMSO is ready for use. The final concentration of the prepared probe is 10 mu M and contains 30 percent of CH₃The CN solution PBS respectively and sodium bisulfite (0-2 mM) with different concentrations fully act, the excitation wavelength is set to 375 nm, fluorescence detection is carried out, the fluorescence intensity in each system is obtained, and the change of the fluorescence intensity along with the concentration of the bisulfite is shown in figure 4 (a); a standard curve of the ratio of fluorescence intensity to bisulfite concentration was established, as shown in FIG. 4 (b). The intensity of fluorescence at 660 nm gradually decreased and the intensity of fluorescence at 460nm gradually increased with increasing bisulfite concentration. When the bisulfite concentration reached 2 mM, the reaction system reached a saturated state.

Example 3 kinetics of response of fluorescent probes to bisulfite

The detection SO of the invention is prepared at a concentration of 1 mM₂The test stock solution of ratiometric fluorescent probe Ph-CN in DMSO was used.A 10 mM sodium bisulfite solution was prepared.20. mu. L probe stock, 580. mu. L CH, was added to the boat₃CN and 400 mu L sodium bisulfite solution, using phosphate buffer solution to fix the volume to 2 m L, mixing uniformly and then carrying out fluorescence detection (lambda)_ex= 375 nm), a plot of the fluorescence intensity ratio versus time was constructed and the results are shown in fig. 5. As can be seen from FIG. 5, the fluorescence intensity did not change after 5s, indicating that the reaction was complete.

Example 4 selectivity of fluorescent probes for different ions

The detection SO of the invention is prepared at a concentration of 1 mM₂The test stock solution of ratiometric fluorescent probe Ph-CN in DMSO was ready for use.A solution of various anions, cations, small molecules and amino acids at a concentration of 10 mM was prepared for use.20. mu. L probe stock, 580. mu. L CH, was added to the boat₃CN and 400 mu L test solution, using phosphate buffer solution to make volume to 2 m L, shaking up and then making fluorescence detection (lambda ex = 375 nm), establishing a bar graph of fluorescence intensity ratio and each ion, and the resultSee fig. 6, wherein the added ions, small molecules and amino acids No. 1-24 are: homocystine, glutathione, sodium thiocyanate, sodium sulfide, glyoxal, cysteine, potassium chloride, magnesium chloride, hydrogen peroxide, sodium fluoride, sodium nitrite, ammonium acetate, sodium thiosulfate, potassium nitrate, calcium chloride, sodium sulfite, benzaldehyde, sodium sulfate, tert-butyl hydroperoxide, potassium iodide, sodium bromide, ammonium phosphate, a probe, sodium bisulfite. From FIG. 6 it can be seen that other ions, small molecules and amino acids have little effect on the response of the probe Ph-CN to bisulfite.

Example 5 response of fluorescent probes to bisulfite at different pH

The detection SO of the invention is prepared at a concentration of 1 mM₂The test stock solution of ratiometric fluorescent probe Ph-CN in DMSO was used.A 10 mM sodium bisulfite solution was prepared.20. mu. L probe stock, 580. mu. L CH, was added to the boat₃CN and 400. mu. L SO₂The solution was diluted to 2 m L with phosphate buffers (pH = 4-10) having different pH, and fluorescence detection was performed after mixing well (λ:. lamda.)_ex= 375 nm), a plot of fluorescence intensity ratio versus solution pH was established and the results are shown in figure 7. From FIG. 7, it can be seen that different pH's have little effect on the fluorescence of the probe Ph-CN, and that the probe response to bisulfite is good in the acidic and neutral ranges.

Example 6 imaging application of fluorescent probes in Living cells

HepG2 cells were seeded at appropriate density into two sterilized 35 mm imaging dishes in CO₂Incubator (temperature 37 ℃, 5% CO)₂) After the cells adhere to the wall, a fluorescent probe Ph-CN is added into the culture dish to ensure that the final concentration of the fluorescent probe Ph-CN is 10 mu M, after the cells are cultured for 30 minutes, the culture medium is discarded, the cells are washed for 3 times by PBS buffer solution, and an imaging experiment is carried out (excitation wavelength: 405 nm, emission band: 425-475 nm and 580-620 nm).

As a control group, a fluorescent probe Ph-CN was added to the other dish so that the final concentration was 10. mu.M, after incubation for 15 minutes, a sodium bisulfite solution was further added so that the final concentration was 2 mM, after incubation for 15 minutes, the medium was discarded, the cells were washed 3 times with PBS buffer, and imaging experiments were performed (excitation wavelength: 405 nm, emission band: 425-.

The results are shown in FIG. 8, which is an image of a cell incubated with a probe Ph-CN for 30 minutes in a1) -a4) HepG2, and a cell incubated with a probe Ph-CN for 15 minutes in b1) -b4) HepG2, and a sodium bisulfite solution for 15 minutes. From this figure, it can be seen that the cells have strong red fluorescence only when the probe is added, and that the red fluorescence in the cells is very significantly reduced and the blue fluorescence is significantly enhanced after the sodium bisulfite solution is added.

Claims

1. A fluorescent probe for detecting sulfur dioxide has a chemical structural formula shown in formula (I):

formula (I).

2. A method of preparing a fluorescent probe according to claim 1, comprising the steps of:

（1）N₂under protection, under the condition of existence of NaOH and KI, heating 2-methoxyphenothiazine and 1-bromobutane in DMSO for reflux reaction, and purifying reaction liquid to obtain a compound 1:

；

(2) at 0 ℃ N₂Under protection, when phosphorus oxychloride and DMF are stirred until a light pink solid appears, adding a DMF solution of a compound 1, heating for reaction, and purifying a reaction solution to obtain a compound 2:

；

(3) dichloromethane solution of compound 2 and AlCl at 0-5 deg.C₃Mixing, reacting at room temperature, and purifying the reaction liquid to obtain a compound 3:

；

（4）N₂under protection, the compound 3 and the p-aminobenzonitrile react in ethanol at room temperature, and the reaction liquid is purified to obtain orange red solid, namely the fluorescent probe.

3. The method according to claim 2, wherein in the step (1), the reaction temperature is 95 ℃; in the step (2), the reaction temperature is 60 ℃.

4. Use of a fluorescent probe according to claim 1 for the preparation of a bisulfite reagent for detection in a solution, cell or organism.