CN116640147A - Fluorescent probe based on tetrahydropyridine ring hemicyanine and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of fluorescent probes, and particularly relates to a fluorescent probe based on tetrahydropyridine ring hemicyanine, and a preparation method and application thereof. In order to solve the problems that the prior hemicyanine derivative has poor water solubility, so that the requirement on the environment is high and the operation is complex, the invention uses nitrogen to replace-CH ₂ The system is introduced with hetero atoms, the water solubility of molecules is improved, so that an additional group is more easily added to a nitrogen part, and the accumulation of the structures is reduced, thereby increasing the fluorescence quantum yield, and the novel tetrahydropyridine ring hemicyanine derivative and the naked eye detection of pH thereof are provided.

Description

Fluorescent probe based on tetrahydropyridine ring hemicyanine and preparation method and application thereof

Technical Field

The invention belongs to the technical field of fluorescent probes, and particularly relates to a fluorescent probe based on tetrahydropyridine ring hemicyanine, a preparation method thereof and application of the fluorescent probe to naked eye detection of pH, which have excellent anti-interference capability and can be an important analysis tool for detecting pH level change in a biological physiological process.

Background

Protons play an important role in physiological processes in living organisms, including cell proliferation, apoptosis, phagocytosis, signaling and ion transport. Intracellular possession of complex physiological processes, various small building block organelles within the cytoplasm have important roles in coordinating and maintaining cell balance. Protons play a role in regulating the pH of organelles during physiological processes in cells, ensuring that organelles remain within a specific pH range during each physiological process. The pH range of five organelles in humans is about 8.0 for mitochondria, 4.5-5.5 for lysosomes, 6.0-6.7 for golgi, and generally about 7.2 for endoplasmic reticulum and cytoplasm. The stabilization of pH is essential for maintaining proper cell operation, and when the pH balance of a certain organelle is disrupted, serious damage may occur, abnormal apoptosis, free radical generation and membrane shrinkage are all associated with the occurrence of serious diseases. Thus, accurate regulation of the intracellular pH or more accurate monitoring of pH fluctuations of specific organelles is critical to the adoption of information about various cellular functions and pathological processes.

The current fluorescent probe detection technology is widely applied in the biological field, and the complete detection mechanism of the fluorescent probe detection technology enables the biological physiological and pathological research process to be visualized, and is more convenient and quick. Although the fluorescent probes are wide in variety and can detect most substances in the physiological process, a part of fluorescent probes have a short fluorescence emission wavelength, cannot reach a near infrared region, cannot well eliminate background interference when applied to biological imaging, and have weak tissue penetrating capability, so that fluorescent signals cannot be completely collected. In addition, whether the level of the biological ions in the cells is normal or not is a key factor influencing the normal operation of the cells, so that the accurate detection of the change of the level of the biological ions penetrating into biological tissues is important to intuitively observe the influence of various biological ions on physiological and pathological processes.

Because the water solubility of most of the existing hemicyanine derivatives is not good, the requirements on the environment are high, and the operation is complex. Therefore, we assume to synthesize a hemicyanine derivative with good water solubility so as to be convenient for being compatible with a living system, and simultaneously realize good optical stability, high response speed and high reversible cycle times.

Disclosure of Invention

The invention aims to solve the problems that the current hemicyanine derivative has poor water solubility, so that the requirement on the environment is high and the operation is more complex, and provides a novel tetrahydropyridine ring hemicyanine derivative for naked eye detection of pH, which utilizes nitrogen to replace-CH ₂ The system is introduced with hetero atoms, so that the water solubility of the molecules is improved, the nitrogen position is easier to add additional groups, and the stacking of the structures is reduced, so that the fluorescence quantum yield is increased. Firstly synthesizing a cyanine structure containing a tetrahydropyridine ring, and then replacing-Cl in the cyanine with resorcinol to obtain a probe He-Cy. The hydroxyl in the structure of the probe He-Cy can be used for detecting the pH, and the hydroxyl protonation or deprotonation effect triggers the ICT effect of the hemicyanine fluorophore when acid and alkali are different, so that different fluorescence changes are generated, and the detection of the pH is realized.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

a fluorescent probe He-Cy based on tetrahydropyridine ring hemicyanine has the structural formula:

a preparation method of a fluorescent probe based on tetrahydropyridine ring hemicyanine comprises the following steps: heating and refluxing the hemicyanine and methyl iodide in acetonitrile solvent to obtain hemicyanine derivative. 1-methyl-4-piperidone reacts with N, N-dimethylformamide and phosphorus oxychloride in a dichloromethane solution, the product is concentrated and dried, then aniline is added into ethanol for reflux reaction to generate piperidone derivatives, after filtering and collecting solids, the solids are melted into ethanol solution in nitrogen atmosphere with the cyanine derivatives, sodium acetate is added for reaction to obtain a probe precursor compound, resorcinol is added after treatment, the resorcinol is dissolved into acetonitrile solution for heating reaction, and the separated and purified tetrahydropyridine ring half-cyanine derivatives He-Cy are obtained.

The method specifically comprises the following steps:

step 1, dissolving 2, 3-trimethyl indole and methyl iodide in acetonitrile, heating and refluxing for reaction under stirring, cooling the reaction liquid to room temperature after the reaction is finished, spin-drying the solvent, and washing to obtain a compound 1;

step 2, dissolving N, N-dimethylformamide in dichloromethane under nitrogen atmosphere, dissolving phosphorus oxychloride in dichloromethane, dropwise adding a dichloromethane solution of the N, N-dimethylformamide into the dichloromethane solution of the phosphorus oxychloride under ice salt bath condition, reacting under stirring, dropwise adding 1-methyl-4-piperidone into a reaction system, reacting under stirring, changing the solution into yellow liquid, heating the obtained reaction liquid, changing the reaction liquid into orange, cooling to room temperature after the reaction is finished, pouring into cold water, vacuum drying, concentrating the liquid to obtain yellow solid, washing and drying to obtain a compound 2;

step 3, dissolving aniline in ethanol under nitrogen atmosphere, dissolving compound 2 and concentrated hydrochloric acid in N, N-dimethylformamide, dropwise adding an ethanol solution of aniline into an N, N-dimethylformamide solution of compound 2 and concentrated hydrochloric acid under ice salt bath condition, stirring for reaction, pouring the mixture into isopropyl ether after the reaction is finished to obtain purple solid precipitate, filtering and collecting the solid, washing and vacuum drying to obtain compound 3;

step 4, under the nitrogen atmosphere, dissolving the compound 1 and the compound 3 in an ethanol solution, then adding sodium acetate, heating, stirring for reaction, cooling to room temperature after the reaction is finished, spin-drying the liquid to obtain a deep blue solid crude product, and separating and purifying the crude product through column chromatography to obtain a compound 4;

and 5, dissolving resorcinol and anhydrous potassium carbonate in acetonitrile solution under the nitrogen atmosphere, stirring for reaction at room temperature, then slowly adding the compound 4 dissolved in acetonitrile, heating for stirring for reaction, cooling to room temperature after the reaction is finished, and spin-drying the liquid to obtain a crude product dark blue-green solid, and separating and purifying the crude product by column chromatography to obtain the probe.

Further, the molar ratio of the 2, 3-trimethylindole to the methyl iodide in the step 1 is 5:1; the temperature of the heating reflux reaction is 85-90 ℃ and the time is 24 hours; spin drying solvent using spin steaming instrument; isopropyl ether was used for the washing 3 times.

Further, in the step 2, the molar ratio of N, N-dimethylformamide, phosphorus oxychloride to 1-methyl-4-piperidone is 5:4:1; the time of the two stirring reactions is 10min; heating the reaction liquid to 70 ℃, wherein the heating time of the reaction liquid is 3 hours; isopropyl ether was used for the washing 2 times.

Further, in the step 3, the molar ratio of the aniline to the compound 2 is 3:1, and the concentrated hydrochloric acid is used in an amount of 2mmol of the compound 2, namely 0.7mL of concentrated hydrochloric acid is used; stirring reaction is carried out for 20min at the temperature of 10-15 ℃; the washing was performed twice with cold distilled water and twice with isopropyl ether.

Further, in the step 4, the molar ratio of the compound 1 to the compound 3 to the sodium acetate is 4:1:9; heating to 70 ℃, and stirring for 4 hours; the eluent for column chromatography separation and purification is dichloromethane/methanol=10:1 (V/V).

Further, the molar ratio of the intermediate diphenol, the anhydrous potassium carbonate and the compound 4 in the step 5 is 2:2:1, stirring reaction time is 0.5h, heating to 50 ℃ and stirring reaction is carried out for 12h, and eluent for column chromatography separation and purification is dichloromethane/methanol=10:1 (V/V).

The application of a fluorescent probe based on tetrahydropyridine ring hemicyanine in detecting pH.

Further, the method for detecting pH comprises the following steps:

(1) Preparing fluorescent probe mother liquor by using dimethyl sulfoxide and a fluorescent probe; weighing Na ₂ HPO ₄ 、KH ₂ PO ₄ Dissolving in ultrapure water to obtain PBS buffer solution, and then using a pH meter to regulate the pH of the solution by dropwise adding NaOH solution and HCl solution to obtain pH solution with the pH value within the range of 3-12;

(3) And respectively adding the prepared pH solution, the fluorescent probe mother solution and the PBS buffer solution into the cuvette to obtain diluted fluorescent probe solution, and placing the cuvette in an ultraviolet-visible absorption spectrometer for testing to obtain ultraviolet-visible absorption spectrograms under different pH conditions, wherein the ultraviolet-visible absorption spectrograms are observed by naked eyes, and the colors of the probe solutions are different when the pH values are different.

Compared with the prior art, the invention has the following advantages:

by substitution of nitrogen for-CH ₂ The introduction of heteroatoms into the system improves the water solubility of the molecule, makes it easier to add additional groups to the nitrogen site, and reduces the build-up of such structures, thereby increasing the fluorescence quantum yield. The fluorescence emission of He-Cy can reach near infrared region, and the biological penetrating power is better. The fluorescence intensity of the probe is gradually enhanced along with the increase of the pH value, and the pH value can be identified by naked eyes. And the fluorescence intensity is not changed greatly in a period of time after the reaction, so that the fluorescent dye has good stability. The invention has excellent specificity in the presence of interfering ions, and good reversibility in pH circulating fluorescence detection, and the probe can be reused in detecting pH.

Drawings

FIG. 1 is a diagram of Compound 4 ¹ H NMR chart;

FIG. 2 is a diagram of a probe He-Cy ¹ H NMR chart;

FIG. 3 is a schematic view ofProbe He-Cy ¹³ C NMR chart;

FIG. 4 mass spectrum of probe He-Cy;

FIG. 5 is an ultraviolet-visible absorption spectrum of a pH probe molecule measuring different pH;

FIG. 6 is a graph showing the comparison of color changes of solutions after pH probe molecules detect different pH;

FIG. 7 is a graph of the fluorescence emission intensity of He-Cy at 710nm under the influence of cations, anions, peroxides and biological amino acids.

Detailed Description

Example 1

A structural formula of a fluorescent probe He-Cy based on tetrahydropyridine ring hemicyanine is as follows:

the preparation method comprises the following steps:

step 1 to a 200mL round bottom flask was added 2, 3-trimethylindole (10.0 mmol,1 eq) iodomethane (20.0 mmol,2 eq) in sequence and dissolved in 50mL acetonitrile and connected to a reflux apparatus. Then, the reaction mixture was refluxed with stirring and heated to 90℃for 24 hours. After the reaction is finished, cooling the reaction liquid to room temperature, spin-drying the solvent of the reaction liquid by using a spin-steaming instrument to obtain a crude product, and washing 3 times by using isopropyl ether to obtain a pink compound 1;

step 2N, N-dimethylformamide (20 mmol,5 eq) was added to Schlenk reaction flask 1 and dissolved in 2mL of methylene chloride solution under nitrogen atmosphere, and phosphorus oxychloride (16 mmol,4 eq) was added to Schlenk reaction flask 2 and dissolved in 2mL of methylene chloride solution. The solution in the reaction flask 1 was added dropwise to the reaction flask 2 under ice-salt bath conditions, and reacted for 10 minutes with stirring. 1-methyl-4-piperidone (4 mmol,1 eq) was then added dropwise to the above reaction flask 2 and reacted for 10min with stirring, the solution turned into a yellow liquid. The resulting reaction solution was heated to 70℃and kept for 3 hours, and the reaction solution became orange. After the reaction is finished, cooling to room temperature, pouring the cooled mixture into cold water (10 mL), connecting a vacuum pump drying device, concentrating the liquid to obtain yellow solid, washing the yellow solid with isopropyl ether for 2 times, and drying to obtain a compound 2;

step 3, aniline (6 mmol,3 eq) was added to Schlenk flask 1 and dissolved in 3mL of ethanol under nitrogen atmosphere, and compound 2 (2 mmol,1 eq) and concentrated hydrochloric acid (0.7 mL) were added to Schlenk flask 2 and dissolved in 2mL of N, N-dimethylformamide. Under the ice salt bath condition, the solution in the reaction bottle 1 is dripped into the reaction bottle 2, and then the reaction temperature is kept between 10 ℃ and 15 ℃ and is stirred for 20min. After the reaction is finished, pouring the mixture into 75mL of isopropyl ether to obtain a purple solid precipitate, filtering and collecting the solid, washing twice with cold distilled water, washing twice with isopropyl ether, and finally drying in vacuum to obtain a purple compound 3;

step 4, under the nitrogen atmosphere, sequentially adding a compound 1 (4 mmol,4 eq) and a compound 3 (1 mmol,1 eq) into a reaction bottle, dissolving the mixture in an ethanol solution (5 mL), then adding sodium acetate (9 mmol,9 eq), heating to 70 ℃, stirring and reacting for 4 hours, cooling to room temperature after the reaction is finished, and spin-drying the liquid by a spin-steaming instrument to obtain a dark blue solid crude product, and separating and purifying the crude product by column chromatography (eluent ratio: dichloromethane/methanol=10:1, silica gel 200-300 meshes) to obtain a compound 4;

1H NMR (600 mhz, chloro-d) delta 7.54 (d, j=2.8 hz, 3H), 7.45 (d, j=8.0 hz, 2H), 7.26 (d, j=7.8 hz, 2H), 7.21 (d, j=7.3 hz, 2H), 7.02 (d, j=8.0 hz, 2H), 5.93 (d, j=14.3 hz, 2H), 4.27 (s, 4H), 3.32 (s, 3H), 3.12 (s, 3H), 1.66 (s, 6H), 1.26 (s, 6H), 0.91-0.81 (m, 3H) 13C NMR (151 mhz, chloro-d) delta 174.05,146.50,143.99,142.68,141.34,128.78,125.63,122.11,110.92,102.98,53.41,52.80,49.39,42.33,33.60,29.68,28.09 [ C32H37ClN3] +,499.2734, found: 499.2738.

step 5, resorcinol (2 mmol,2 eq) and anhydrous potassium carbonate (2 mmol,2 eq) were added sequentially to a reaction flask under nitrogen atmosphere, dissolved in 5mL of acetonitrile solution, and reacted for 0.5h at room temperature under stirring. Then, compound 4 (1 mmol,1 eq) dissolved in 5mL acetonitrile solution is injected into a reaction bottle by a needle tube, heated to 50 ℃, stirred and reacted for 12 hours, cooled to room temperature after the reaction is finished, and the liquid is dried by a rotary evaporator to obtain a dark blue-green solid crude product, and the crude product is separated and purified by column chromatography (eluent ratio: dichloromethane/methanol=10:1, silica gel 200-300 meshes) to obtain the probe He-Cy.

Example 2

The fluorescent probe is applied to detecting pH.

The method for detecting the pH by the fluorescent probe comprises the following steps:

(1) Preparing a fluorescent probe mother solution of 2mM by using DMSO; weigh 4.25g of Na ₂ HPO ₄ KH of 4.08g ₂ PO ₄ Dissolved in 600mL of ultrapure water to give a buffer solution PBS having a concentration of 0.1M. Then using a pH meter, and regulating the pH of the solution by dropwise adding a proper amount of 0.1M NaOH solution and 0.1M HCl solution to obtain a pH solution with the pH value in the range of 3-12;

(2) He-Cy mother liquor was diluted with buffer PBS. To the cuvette, 10. Mu.L of the prepared pH solution and 1950. Mu.L of the probe mother solution 40. Mu. L, PBS solution were added, respectively, to obtain a He-Cy solution having a final concentration of 20. Mu.M, and the cuvette was subjected to a test in an ultraviolet-visible absorption spectrometer to obtain an ultraviolet-visible absorption spectrum under different pH conditions (as shown in FIG. 5).

As can be seen by the naked eye, the color of the probe solution is different when the pH value is different, and as shown in fig. 6, the color of the solution is dark blue when the ph=3.60, the solution is gradually changed from dark purple to blue when the ph=4.50-7.12, and the bright blue of the solution is gradually weakened when the ph=8.00-12.00.

Example 3

Preparing mother liquor of tetrahydropyridine ring hemicyanine probe: 5.26mg of probe He-Cy was weighed out, dissolved in a DMSO solvent, and fixed to a volume of 10.0mL in a volumetric flask to obtain a He-Cy mother solution having a concentration of 1.0 mM.

Under exactly the same conditions as the pH to be measured, cations, anions, peroxides and biogenic amino acids were added thereto, and it is apparent from FIG. 7 that the cations (Na ⁺ ，K ⁺ ，Mg ²⁺ ，Ca ²⁺ ，Cu ²⁺ ) Anions (Cl) ^- ，I ^- ，SO ₄ ^2- ) Biological amino acids (glutathione, L-leucine, D-tryptophan), oxides (H ₂ O ₂ ) For probe He-Cy at 71The fluorescence emission intensity at 0nm has little effect. This shows that the probe He-Cy has strong specific recognition capability, is not interfered by the cations, anions, peroxides and biological amino acids, and has good selectivity and sensitivity for detecting the pH of the buffer solution when cell imaging is carried out.

Claims (9)

1. The fluorescent probe based on the tetrahydropyridine ring hemicyanine is characterized by comprising the following structural formula:

2. a method for preparing a tetrahydropyridine ring hemicyanine-based fluorescent probe as claimed in claim 1, comprising the steps of:

step 1, dissolving 2, 3-trimethyl indole and methyl iodide in acetonitrile, heating and refluxing for reaction under stirring, cooling the reaction liquid to room temperature after the reaction is finished, spin-drying the solvent, and washing to obtain a compound 1;

step 2, dissolving N, N-dimethylformamide in dichloromethane under nitrogen atmosphere, dissolving phosphorus oxychloride in dichloromethane, dropwise adding a dichloromethane solution of the N, N-dimethylformamide into the dichloromethane solution of the phosphorus oxychloride under ice salt bath condition, reacting under stirring, dropwise adding 1-methyl-4-piperidone into a reaction system, reacting under stirring, changing the solution into yellow liquid, heating the obtained reaction liquid, changing the reaction liquid into orange, cooling to room temperature after the reaction is finished, pouring into cold water, vacuum drying, concentrating the liquid to obtain yellow solid, washing and drying to obtain a compound 2;

step 3, dissolving aniline in ethanol under nitrogen atmosphere, dissolving compound 2 and concentrated hydrochloric acid in N, N-dimethylformamide, dropwise adding an ethanol solution of aniline into an N, N-dimethylformamide solution of compound 2 and concentrated hydrochloric acid under ice salt bath condition, stirring for reaction, pouring the mixture into isopropyl ether after the reaction is finished to obtain purple solid precipitate, filtering and collecting the solid, washing and vacuum drying to obtain compound 3;

step 4, under the nitrogen atmosphere, dissolving the compound 1 and the compound 3 in an ethanol solution, then adding sodium acetate, heating, stirring for reaction, cooling to room temperature after the reaction is finished, spin-drying the liquid to obtain a deep blue solid crude product, and separating and purifying the crude product through column chromatography to obtain a compound 4;

and 5, dissolving resorcinol and anhydrous potassium carbonate in acetonitrile solution under the nitrogen atmosphere, stirring for reaction at room temperature, then slowly adding the compound 4 dissolved in acetonitrile, heating for stirring for reaction, cooling to room temperature after the reaction is finished, and spin-drying the liquid to obtain a dark blue-green solid crude product, and separating and purifying the crude product by column chromatography to obtain the probe.

3. The method for preparing a fluorescent probe based on tetrahydropyridine ring hemicyanine according to claim 2, wherein the molar ratio of 2, 3-trimethylindole to methyl iodide in the step 1 is 5:1; the temperature of the heating reflux reaction is 85-90 ℃ and the time is 24 hours; spin drying solvent using spin steaming instrument; isopropyl ether was used for the washing 3 times.

4. The method for preparing the fluorescent probe based on the tetrahydropyridine ring half cyanine according to claim 2, wherein the molar ratio of N, N-dimethylformamide, phosphorus oxychloride to 1-methyl-4-piperidone in the step 2 is 5:4:1; the time of the two stirring reactions is 10min; heating the reaction liquid to 70 ℃, wherein the heating time of the reaction liquid is 3 hours; isopropyl ether was used for the washing 2 times.

5. The method for preparing the fluorescent probe based on the tetrahydropyridine ring half cyanine according to claim 2, wherein in the step 3, the molar ratio of aniline to the compound 2 is 3:1, and the amount of concentrated hydrochloric acid is 2mmol of the compound 2, and 0.7mL of concentrated hydrochloric acid is used; stirring reaction is carried out for 20min at the temperature of 10-15 ℃; the washing was performed twice with cold distilled water and twice with isopropyl ether.

6. The method for preparing a fluorescent probe based on tetrahydropyridine ring hemicyanine according to claim 2, wherein the molar ratio of compound 1, compound 3 to sodium acetate in step 4 is 4:1:9; heating to 70 ℃, and stirring for 4 hours; the eluent for column chromatography separation and purification is dichloromethane/methanol=10:1 (V/V).

7. The method for preparing the fluorescent probe based on the tetrahydropyridine ring half cyanine according to claim 2, wherein the molar ratio of the intermediate diphenol, the anhydrous potassium carbonate and the compound 4 in the step 5 is 2:2:1, stirring reaction time is 0.5h, heating to 50 ℃ and stirring reaction is carried out for 12h, and eluent for column chromatography separation and purification is dichloromethane/methanol=10:1 (V/V).

8. Use of a tetrahydropyridine ring hemicyanine based fluorescent probe as claimed in claim 1, in the detection of pH.

9. Use of a fluorescent probe based on tetrahydropyridine ring hemi-cyanine according to claim 8, wherein the method of detecting pH comprises the steps of:

(1) Preparing fluorescent probe mother liquor by using dimethyl sulfoxide and a fluorescent probe; weighing Na ₂ HPO ₄ 、KH ₂ PO ₄ Dissolving in ultrapure water to obtain PBS buffer solution, and then using a pH meter to regulate the pH of the solution by dropwise adding NaOH solution and HCl solution to obtain pH solution with the pH value within the range of 3-12;

(3) And respectively adding the prepared pH solution, the fluorescent probe mother solution and the PBS buffer solution into the cuvette to obtain diluted fluorescent probe solution, and placing the cuvette in an ultraviolet-visible absorption spectrometer for testing to obtain ultraviolet-visible absorption spectrograms under different pH conditions, wherein the ultraviolet-visible absorption spectrograms are observed by naked eyes, and the colors of the probe solutions are different when the pH values are different.