CN113735762B

CN113735762B - Water-soluble fluorescent probe with aggregation-induced emission characteristic and preparation method and application thereof

Info

Publication number: CN113735762B
Application number: CN202111009124.2A
Authority: CN
Inventors: 陈杜刚; 冯杨振; 闫志国; 余响林; 李婉青; 梁文杰
Original assignee: Wuhan Institute of Technology
Current assignee: Wuhan Institute of Technology
Priority date: 2021-08-31
Filing date: 2021-08-31
Publication date: 2022-07-19
Anticipated expiration: 2041-08-31
Also published as: CN113735762A

Abstract

The invention relates to a water-soluble fluorescent probe with aggregation-induced emission characteristics, and a preparation method and application thereof. Firstly, a compound 1 and 4-pyridine boronic acid pinacol ester are subjected to catalytic reaction in an organic solvent at 65-120 ℃ to obtain a compound 2, the compound 2 and iodomethane are subjected to reaction in the organic solvent at 25-80 ℃ to obtain a compound 3, and the compound 3 and boron tribromide are subjected to reaction in the organic solvent at 20-40 ℃ to obtain a target product. The method has the advantages of easily obtained raw materials, simple synthesis process, high yield and the like, and experiments prove that the prepared fluorescent probe can be used for monitoring the viscosity in mitochondria and has the advantages of high selectivity, high sensitivity response, high anti-interference performance, high imaging contrast ratio and the like.

Description

Water-soluble fluorescent probe with aggregation-induced emission characteristic and preparation method and application thereof

Technical Field

The invention relates to the technical field of organic small-molecule fluorescent probes, in particular to a water-soluble fluorescent probe with aggregation-induced emission characteristics and a preparation method and application thereof.

Background

Mitochondria are the motor chamber of cells and play an important role in signal transduction, cell differentiation, apoptosis, calcium regulation, etc., and these functions are closely related to fluctuations in mitochondrial viscosity. Changes in mitochondrial viscosity can further affect many cellular processes of physiological activity, such as membrane fusion, metabolite diffusion, protein aggregation, signal transduction, and the like. It has been reported that intracellular viscosity abnormality is often associated with diseases such as alzheimer's disease, diabetes, atherosclerosis, and hypertension, and thus some diseases can be effectively diagnosed by detecting intracellular viscosity. However, due to the complexity of the intracellular environment, the selective real-time in situ detection of viscosity remains a significant challenge. Therefore, the development of a probe tool which has targeting effect on mitochondria and can specifically respond to viscosity is very significant, and the probe tool provides a basis for revealing the chemical and biological effects of viscosity in related diseases.

The traditional viscosity measurement means mainly comprise a capillary viscometer, a rotary viscometer and a falling ball viscometer, and the methods can only be used in the macroscopic field and cannot monitor the viscosity at the cellular level. The fluorescent probe has the advantages of high sensitivity, good time-space resolution, visualization, nondestructive detection and the like, and is widely used for selective imaging of various components in a living body. Through rational molecular design, probe tools can be developed that selectively respond to intracellular viscosity, even within specific organelles.

In recent years, fluorescent molecules with Aggregation Induced Emission (AIE) characteristics have received much attention due to their excellent light stability, large stokes shift, and high imaging contrast. Based on the mechanism of intramolecular motion limitation, when the intracellular viscosity is increased, the non-radiative transitions such as rotation and vibration in molecules can be inhibited, so that the fluorescence enhancement of the molecules realizes the response to the viscosity. However, conventional AIE-type fluorescent probes are hydrophobic and aggregation in physiological environments also leads to increased fluorescence, which can severely interfere with viscosity detection. Therefore, it is important to design AIE molecules that have excellent water solubility in a cellular environment, are not affected by aggregation, and specifically respond to viscosity only, and to use them for viscosity monitoring.

The retrieval shows that Chinese patent CN108516950B discloses targeted mitochondrial viscosity fluorescent probes Mito-AIE1 and Mito-AIE2 based on tetraphenylethylene, and the scheme solves the water solubility problem of AIE type probes, but still has the problems of weak anti-interference capability, insufficient detection sensitivity and the like. Analysis shows that the fluorescent probe molecule connects a strong receptor pyridinium or indolyl salt group with tetraphenyl ethylene through a carbon-carbon double bond, and the double bond is easy to react with active oxygen (such as hypochlorous acid) and a nucleophilic reagent (such as sulfur ions) to cause the fluorescence of the probe to change, so that the fluorescent probe is easily interfered by the active oxygen and the nucleophilic component when applied to cells. When the viscosity of the solution was increased from 1.4cP to 563.6cP, the fluorescence turn-on times of the two probes were 38 times and 6 times, respectively, and there was a large room for improvement.

Aiming at the problems of weak anti-interference capability and low imaging sensitivity of the water-soluble AIE type viscosity fluorescent probe in the prior art, the invention develops a fluorescent probe with a novel molecular structure.

Disclosure of Invention

One of the objectives of the present invention is to provide a water-soluble fluorescent probe with aggregation-induced emission characteristics, the molecular structure of which is shown in formula (I):

the second objective of the present invention is to provide a method for preparing the above water-soluble fluorescent probe with aggregation-induced emission characteristics, which comprises the following steps:

(a) reacting the compound 1 with 4-pyridine boronic acid pinacol ester in the presence of an organic solvent I, a catalyst I and an additive I to obtain a compound 2

(b) Reacting the compound 2 with methyl iodide in the presence of an organic solvent II to obtain a compound 3

(c) Reacting the compound 3 with boron tribromide in the presence of an organic solvent III to obtain a compound of a formula (I)

Further, the organic solvent I in the step (a) is at least one selected from toluene, tetrahydrofuran, methanol, ethanol, dichloromethane, 1, 4-dioxane and N, N-dimethylformamide, and is preferably tetrahydrofuran; the catalyst I is at least one selected from tetrakis (triphenylphosphine) palladium, bis (triphenylphosphine) palladium chloride and [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride, and is preferably tetrakis (triphenylphosphine) palladium; the additive I is at least one selected from potassium carbonate aqueous solution, sodium carbonate aqueous solution, potassium phosphate aqueous solution and tetraethylammonium hydroxide aqueous solution, and is preferably potassium carbonate aqueous solution. The additive needed by the reaction is alkaline solution, and the additive needs to be matched with a palladium catalyst for use, so that the reaction can be smoothly carried out.

Further, the mol ratio of the compound 1 to the 4-pyridine boronic acid pinacol ester, the catalyst I and the additive I in the step (a) is 1 (1-2): (0.01-0.1): (3-10).

Further, in the step (a), under the protective atmosphere, dissolving the compound 1 and 4-pyridine boronic acid pinacol ester in an organic solvent I, adding the catalyst I and the additive I, uniformly stirring, heating the obtained mixture to 65-120 ℃, stirring, reacting for 12-24h, finally extracting and separating to obtain an organic phase, drying the organic phase, and separating by silica gel column chromatography to obtain the compound 2, wherein an eluent is petroleum ether/ethyl acetate (v/v, 4/1).

Further, the organic solvent II in the step (b) is at least one selected from tetrahydrofuran, 1, 4-dioxane, ethanol, methanol, acetonitrile and N, N-dimethylformamide, and is preferably N, N-dimethylformamide.

Further, the molar ratio of compound 2 to methyl iodide in step (b) is 1: (1-5).

Further, in the step (b), the compound 2 is dissolved in an organic solvent II under the protective atmosphere, methyl iodide is slowly added, then the temperature is raised to 25-80 ℃ in the dark, the reaction is stirred for 2-6h, and finally the compound 3 is obtained by silica gel column chromatography separation, wherein an eluent is dichloromethane/methanol (v/v, 15/1).

Further, the organic solvent III in the step (c) is at least one selected from dichloromethane, chloroform, acetonitrile, tetrahydrofuran and toluene, and is preferably dichloromethane.

Further, the molar ratio of compound 3 to boron tribromide in step (c) is 1: (1-5).

Further, in the step (c), respectively dissolving the compound 3 and boron tribromide in the organic solvent III under a protective atmosphere to obtain a compound 3 solution and a boron tribromide solution; and (3) putting the compound 3 solution in an ice-water bath, adding a boron tribromide solution into the ice-water bath, heating the obtained mixture to 20-40 ℃, stirring and reacting for 12-24h, and finally recrystallizing and purifying to separate the compound shown in the formula (I), wherein a solvent used for recrystallization is dichloromethane/n-hexane (v/v, 2/1).

The third purpose of the present invention is to provide an application of the above water-soluble fluorescent probe in monitoring viscosity change in cells (especially in mitochondria) by means of fluorescence imaging.

The water-soluble fluorescent probe molecule is based on a tetraphenyl ethylene framework and has a D-pi-A type structure with phenolic hydroxyl as an electron donor (D) and pyridinium as an electron acceptor (A). Compared with the prior art represented by CN108516950B, the molecular design strategy of the invention has the following advantages: (1) the water solubility is good, the molecular structure is simpler, the synthesis is easier, and the total yield is higher; (2) the receptor pyridinium is connected into molecules through single bonds instead of common carbon-carbon double bonds, so that the damage of nucleophilic components and active oxygen components in a biological environment to the molecular structure of the probe is thoroughly avoided, and the anti-interference capability of the probe is greatly improved; (3) when the solution viscosity increased from 1.4cP to 563.6cP, the fluorescence increased 128-fold and the imaging sensitivity increased significantly.

In conclusion, the invention simultaneously and reasonably constructs probe molecules from multiple aspects of fluorescent group, water solubility, chemical stability, viscosity response and the like, applies the probe molecules to fluorescence imaging of viscosity in mitochondria, and has the advantages of high imaging contrast, strong anti-interference capability, high sensitivity and the like.

Drawings

FIG. 1 is a nuclear magnetic hydrogen spectrum of the compound of formula (I) in example 1.

FIG. 2 is a mass spectrum of the compound of formula (I) in example 1.

FIG. 3 is a fluorescence spectrum of the compound of formula (I) according to the change of solution viscosity in example 1.

FIG. 4 is a graph showing the solubility test of the compound of formula (I) in phosphate buffer solution in example 1.

FIG. 5 is a test of the anti-interference ability of the compound of formula (I) in example I.

FIG. 6 is a graph showing the staining of mitochondria in the co-localization experiment of the compound of formula (I) in example 1.

FIG. 7 is a graph of the cytographic image of the compound of formula (I) on viscosity in example 1.

Detailed Description

In order to make those skilled in the art fully understand the technical solutions and advantages of the present invention, the following description is further provided with reference to the specific embodiments and the accompanying drawings.

All the raw materials of the invention are generally sold in the market without special instructions, and related biological experiments strictly comply with the requirements of relevant laws and regulations.

Example 1

The reaction procedure for synthesizing the target fluorescent probe of formula (I) in this example is shown in the following figure

(1) Synthesis of Compound 2

Compound 1(1.50g, 3.40mmol), 4-pyridineboronic acid pinacol ester (0.84g, 4.08mmol) were dissolved in dry tetrahydrofuran (30mL) under argon protection, and tetrakis (triphenylphosphine) palladium (in an amount of 5% of the compound 1 mol) and K were added₂CO₃Aqueous solution (8mL, 2M). The mixture is heated to 70 ℃ and then stirred and reacted for 20h under the condition of heat preservation. Cooling to room temperature after reaction, adding water for extraction and liquid separation, collecting an organic phase, and using anhydrous Na₂SO₄After drying and concentration, the residue was purified by silica gel column chromatography using petroleum ether/ethyl acetate (v/v, 4/1) as eluent to give compound 2(1.08g) in 73% yield.

(2) Synthesis of Compound 3

Compound 2(1.00g, 2.28mmol), methyl iodide (0.97g, 6.84mmol) were dissolved in dry acetonitrile (15mL) under argon. The mixture is heated to 80 ℃ under the condition of keeping out of the light and stirred for reaction for 6 hours. After the reaction was completed, cooled to room temperature, directly concentrated and purified by silica gel column chromatography using dichloromethane/methanol (v/v, 15/1) as an eluent, to give compound 3 (1.17g) in 89% yield.

(3) Synthesis of Compounds of formula (I)

Compound 3(0.50g, 0.85mmol) was dissolved in dichloromethane (30mL) and boron tribromide (0.86g, 3.40mmol) was dissolved in dichloromethane (10mL) under argon. Putting the dichloromethane solution of the compound 3 into an ice water bath, slowly dropping the dichloromethane solution of boron tribromide, and stirring and reacting at room temperature (25 ℃) for 12 hours. After completion of the reaction, the mixture was directly concentrated and recrystallized from methylene chloride/n-hexane (v/v, 2/1) to obtain the compound of the formula (I) (0.27g) in a yield of 46%.

The nuclear magnetic hydrogen spectrum and the mass spectrum of the compound of the formula (I) are respectively shown in figures 1-2. The spectrum results are as follows:¹H NMR(400MHz,DMSO)δ[ppm]:9.46(d,J＝12Hz,1H),8.95(t, J＝12Hz,2H),8.43(dd,J＝16Hz,2H),7.90(dd,J＝28Hz,2H), 7.25-7.08(m,8H),7.07-6.94(m,4H),6.82(d,J＝8Hz,1H),6.76(d,J＝8 Hz,1H),6.54(dd,J＝24Hz,2H),4.30(d,J＝4Hz,3H)。HRMS for C₃₂H₂₆NO⁺m/z:[M-I^-]⁺440.2009 for Calcd; found: 440.2002. The nuclear magnetic hydrogen spectrum, carbon spectrum and mass spectrum data confirm that the target product with the structure of the formula (I) is really synthesized according to the method.

Examples 2-6 were carried out by varying the reaction conditions (as described in table 1) with reference to the procedure of example 1.

TABLE 1 comparison of results of different reaction conditions in examples 2-6

To further understand the performance of the fluorescent probe with the structure of formula (I) prepared by the present invention, the following experiment was performed by taking the product of example 1 as an example.

(1) Viscosity response test, water solubility test and anti-interference capability test of fluorescent probe

The fluorescent probe prepared in example 1 was dissolved in dimethyl sulfoxide to prepare a 1mM test stock solution for use. The change in the viscosity of the solvent was simulated with phosphate buffered saline (PBS, pH 7.40) of different glycerol contents, which were: 0, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 99%. The probe mother solution was added to solvents of different viscosities to prepare a test solution having a probe concentration of 10. mu.M. The fluorescence spectra of the test solutions were measured, and the results are shown in FIG. 3. As can be seen from FIG. 3, the fluorescence intensity of the probe gradually increased with the increase in the viscosity of the solvent, and the logarithmic value of the fluorescence intensity at 611nm linearly correlated with the logarithmic value of the viscosity of the solvent.

The fluorescence spectra of the probes at different concentrations were tested in PBS and the results are shown in FIG. 4. The linear relationship between the fluorescence intensity and the concentration of the probe in the range of 0-40. mu.M indicates that the probe is very water-soluble in PBS and does not aggregate.

To the PBS/glycerol (v/v, 4/6) mixed solution of the probe, each interfering component (SO) was added₃ ^2-、S^2-、GSH、H₂O₂、ONOO^-、ClO^-) Then, the fluorescence spectrum of the probe was measured, and the result is shown in fig. 5 (1: control, non-interfering components; 2: SO₃ ^2-；3：S^2-；4： GSH；5：H₂O₂；6：ONOO^-；7：ClO^-). As can be seen from the figure, none of these components had an effect on the fluorescence of the probe.

(2) Mitochondrial co-localization imaging of fluorescent probes

Taking human normal hepatocyte LO with appropriate density₂The cells were inoculated into a sterilized culture dish, and co-staining experiments were performed on the cells with the nuclear dye DAPI (5. mu.M), the fluorescent probe (10. mu.M), and the commercialized mitochondrial stain Mito-Tracker Green (5. mu.M), respectively, and then observed under a confocal laser scanning microscope, with the results shown in FIG. 6.

In FIG. 6, A is an image of a blue channel cell, and only the cell nucleus is stained, indicating that the cell is in good condition; b is a cell imaging diagram of Mito-Tracker Green in a Green channel; c is a cell imaging diagram of the fluorescent probe in a red channel; d is an overlay of A, B, C. As can be seen from FIG. 6, the red fluorescence of the probe highly coincides with the Green fluorescence of Mito-Tracker Green, which indicates that the fluorescent probe prepared in this example 1 can be precisely located in the mitochondria of cells, and can be used to detect viscosity changes in mitochondria.

(3) Cellular imaging of viscosity by fluorescent probes of formula I

Taking human normal liver cell LO with proper density₂Inoculated into sterilized petri dishes and randomly divided into 5 groups. The first group is a control group and is not processed; the second group was incubated with fluorescent probe (10. mu.M) for 30 min; the third group is cultured with Monensin (Monensin, 10 μ M) for 60min, and then incubated with fluorescent probe (10 μ M) for 30 min; fourthly, adding Nystatin (10 μ M) for culturing for 60min, and adding fluorescent probe (10 μ M) for incubation for 30 min; in the fifth group, 3% alcohol was added for 60min, and then a fluorescent probe (10. mu.M) was added for incubation for 30 min. The 5 groups of cells were individually placed under a confocal laser microscope to observe imaging, and the results are shown in FIG. 7.

As can be seen in fig. 7, cells incubated with the probe alone emitted weak fluorescence; cells were cultured with monensin, nystatin, and ethanol to stimulate an increase in viscosity in mitochondria, and then cultured with a fluorescent probe to emit intense red fluorescence. This indicates that the fluorescent probe prepared in example 1 can be used to detect a change in intracellular viscosity.

Claims

1. A water-soluble fluorescent probe having aggregation-induced emission characteristics, comprising: the molecular structure of the fluorescent probe is shown as the formula (I):

2. the method for preparing a fluorescent probe according to claim 1, characterized in that the method comprises the steps of:

The organic solvent I in the step (a) is selected from at least one of toluene, tetrahydrofuran, methanol, ethanol, dichloromethane, 1, 4-dioxane and N, N-dimethylformamide, the catalyst I is selected from at least one of tetrakis (triphenylphosphine) palladium, bis (triphenylphosphine) palladium chloride and [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride, the additive I is selected from at least one of potassium carbonate aqueous solution, sodium carbonate aqueous solution, potassium phosphate aqueous solution and tetraethylammonium hydroxide aqueous solution, the organic solvent II in the step (b) is selected from at least one of tetrahydrofuran, 1, 4-dioxane, ethanol, methanol, acetonitrile and N, N-dimethylformamide, and the organic solvent III in the step (c) is selected from dichloromethane, chloroform, acetonitrile, tetrahydrofuran, and N, N-dimethylformamide, At least one of toluene.

3. The method of claim 2, wherein: the organic solvent I in the step (a) is tetrahydrofuran, the catalyst I is tetrakis (triphenylphosphine) palladium, the additive I is potassium carbonate aqueous solution, the organic solvent II in the step (b) is N, N-dimethylformamide, and the organic solvent III in the step (c) is dichloromethane.

4. The method of claim 2, wherein: the mol ratio of the compound 1 to the 4-pyridine boronic acid pinacol ester, the catalyst I and the additive I in the step (a) is 1 (1-2): (0.01-0.1): (3-10).

5. The method of claim 2, wherein: dissolving a compound 1 and 4-pyridine boronic acid pinacol ester in an organic solvent I under a protective atmosphere, adding a catalyst I and an additive I, uniformly stirring, heating the obtained mixture to 65-120 ℃, fully stirring for reaction, finally extracting and separating liquid to obtain an organic phase, drying the organic phase, and separating by silica gel column chromatography to obtain a compound 2, wherein an eluent is petroleum ether/ethyl acetate.

6. The method of claim 2, wherein: the molar ratio of the compound 2 to the methyl iodide in the step (b) is 1: (1-5).

7. The method of claim 2, wherein: dissolving the compound 2 in an organic solvent II under the protective atmosphere in the step (b), slowly adding methyl iodide, heating to 25-80 ℃ in the dark, fully stirring for reaction, and finally separating by silica gel column chromatography to obtain a compound 3, wherein an eluent is dichloromethane/methanol.

8. The method of claim 2, wherein: the molar ratio of the compound 3 to the boron tribromide in the step (c) is 1: (1-5).

9. The method of claim 2, wherein: respectively dissolving the compound 3 and boron tribromide in an organic solvent III under a protective atmosphere in the step (c) to obtain a compound 3 solution and a boron tribromide solution; and (3) putting the compound 3 solution in an ice-water bath, adding a boron tribromide solution into the ice-water bath, heating the obtained mixture to 20-40 ℃, fully stirring the mixture for reaction, and finally, purifying and separating the compound shown in the formula (I) through recrystallization, wherein a solvent used for recrystallization is dichloromethane/n-hexane.

10. Use of the fluorescent probe of claim 1 for monitoring changes in intracellular viscosity by means of fluorescence imaging.