CN114177175B - Application of hydrophilic four-cation ring based on TPE construction - Google Patents

Abstract

The invention relates to an application of hydrophilic TPE-based tetracationic ring in NADPH recognition and cell imaging. The material takes TPE and 4,4' -bipyridine units as construction units. TPE-cyc is able to specifically recognize NADPH and complex it in a 1:1 fashion due to electrostatic interactions and pi-pi stacking. After TPE-cyc is internalized by tumor cells, intracellular NADPH can be captured, and NADPH generation reaction NADH+NADP can be broken ⁺ →NADPH+NAD ⁺ Thereby breaking the balance of NADPH participating in the redox process, improving antioxidant capacity to scavenge ROS. Meanwhile, the high concentration of GSH in the tumor cells can reduce the 4,4' -bipyridine (MV) units of TPE-cyc to a free radical cation state, and destroy the Photoinduced Electron Transfer (PET) effect between the electron-rich TPE and electron-deficient bipyridine units, thereby restoring the fluorescence of the TPE units and lighting the tumor cells. Thus, TPE-cyc acts as a GSH responsive fluorescent switch, displaying image cells at high resolution.

Description

Application of hydrophilic four-cation ring based on TPE construction

Technical Field

The invention relates to an application of a hydrophilic tetracationic ring based on TPE construction in biomolecule recognition.

Background

Understanding the importance of molecular recognition in complex biological processes involving various nucleic acids, enzymes, and nucleotides helps chemists develop intelligent structures with specific responses. In recent years, artificial molecular receptors have received increasing attention for their potential use in the medical and biological fields. Nicotinamide Adenine Dinucleotide Phosphate (NADPH), a major cell reductant, plays an important role in maintaining the reduced form of glutathione, GSH. GSH can eliminate Reactive Oxygen Species (ROS) in cells, thereby preventing oxidative damage to cells. Although possible mechanisms of NADPH involvement in physiological processes have been proposed, due to the complexity and uncertainty of existing mechanisms, more extensive research into the mechanisms is still needed. Therefore, it is important to construct a molecular recognition system capable of selectively recognizing NADPH.

The discovery of crown ethers opens the way for supermolecular chemists to design acceptor macrocyclic molecules based on non-covalent interactions or weak coordination. At present, chemists have constructed a wide variety of macrocyclic entities such as cyclodextrins, cucurbiturils, calixarenes, cycloguaranties, and pillar arenes. Stoddart et al found a "blue box" opening a new era of cationic cycloguaranty. The cation ring has multi-cation state and self-assembly with the object rich in pi electrons, so that the cation ring has great potential in molecular recognition. For example, a tetracationic cyclic compound constructed based on pi-electron deficient 4,4' -bipyridine units may selectively complex with pi-electron rich hosts to form a 1:1 or 1:2 complex. Tetraphenyl ethylene (TPE) derivatives are a typical aggregation-induced emission (AIE) luminophore and have been widely used in recent years to build macrocyclic compounds. The cyclic compound constructed based on TPE not only shows excellent photophysical properties, but also has flexible and diverse cavity structures, which can be used to capture biomolecules, because of its AIE effect and propeller structure. Although many TPE-based cationic ring systems have been used for host-guest recognition, there are few examples of hydrophilic TPE cationic ring systems that recognize biomolecules in aqueous media.

The invention discloses a hydrophilic TPE tetra-cationic ring for identifying biomolecules in an aqueous medium.

Disclosure of Invention

The invention aims to provide application of a hydrophilic TPE tetra-cation ring based on TPE construction in NADPH recognition and cell imaging. The TPE tetra-cationic ring (TPE-cyc) is capable of specifically recognizing NADPH and forming a 1:1 host-guest complex. TPE-cyc is not only easy to be phagocytosed by tumor cells, but also can selectively capture intracellular NADPH, thereby breaking the balance of redox processes participated by NADPH and improving the oxidation resistance of cells. Meanwhile, the high concentration of GSH in the tumor cells can reduce the 4,4' -bipyridine (MV) units of TPE-cyc to a free radical cation state and destroy the Photoinduced Electron Transfer (PET) effect between the electron-rich TPE and electron-deficient bipyridine units, so that the fluorescence of the TPE units is recovered and the tumor cells are lightened, and therefore, the TPE-cyc can be used as a GSH-responsive fluorescent switch to display the image cells with high resolution.

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

in a first aspect, the invention provides an application of a hydrophilic TPE tetra-cationic ring shown in a formula (I) in preparing a medicament for inducing cell death,

the hydrophilic TPE tetracationic ring represented by formula (I) induces cell death by capturing NADPH.

Preferably, the cells are tumor cells, further preferably U87 cells or HeLa cells.

Further, in the case of in vitro applications, the application is: the cells are incubated in a cell culture medium containing 4-10. Mu.M, preferably 4. Mu.M, of a hydrophilic TPE tetracationic ring of formula (I) for 24-48h, preferably 24h, to induce cell death.

The molecule also has potential for in vivo use.

In a second aspect, the invention provides an application of hydrophilic TPE tetracationic ring in high-resolution fluorescence imaging of cytoplasm of cells, wherein the application is shown in a formula (I),

the hydrophilic TPE tetracationic ring shown in the formula (I) can capture glutathione in cells, and all cells contain the glutathione, but the concentration of the glutathione in tumor cells is higher, so the invention recommends that the cells are tumor cells.

Preferably, the cells are HeLa cells.

Further, in the case of in vitro applications, the application is: the cells were incubated in a cell culture medium containing 2-4. Mu.M (preferably 2. Mu.M) of a hydrophilic TPE tetracationic ring of formula (I) for 2-8h (preferably 8 h), i.e.blue fluorescence was observed under a high resolution fluorescence imager (excitation wavelength of 350-370 nm).

The above incubations were all performed under common cell culture conditions.

In addition, the invention provides a preparation method of the hydrophilic TPE tetra-cationic ring shown in the formula (I), which comprises the following steps:

(1) Synthesis of intermediate 3: at N ₂ N-butyllithium was first added to the THF solution of compound 1 under an atmosphere, and the resulting orange-red solution was stirred at 0 ℃. Then, a THF solution of compound 2 was added dropwise to the above mixed solution, and after the addition was completed, the mixture was warmed to room temperature and stirred for 6 hours. The reaction was quenched with saturated aqueous ammonium chloride. Extraction was performed with DCM. Anhydrous Na for organic phase ₂ SO ₄ And (5) drying. The crude product obtained is dissolved in toluene and a catalytic amount of p-toluene sulphonic acid is added, using a catalyst system comprising

The molecular sieve dehydration device separates the generated water. NaHCO for toluene layer ₃ Washing with an aqueous solution, washing with anhydrous Na ₂ SO ₄ And (5) drying. The toluene solvent was removed by rotary column chromatography and the crude product was purified by column chromatography.

(2) Synthesis of intermediate compound 4: at N ₂ Under the atmosphere, compound 3 was first dissolved in CCl ₄ Then NBS and dibenzoyl peroxide were added and finally the mixture was heated to 70℃under reflux for 12h.

(3) Synthesis of intermediate compound 5: first, 4' -bipyridine was dissolved in acetonitrile and refluxed at 75 ℃. Compound 4 was then dissolved in acetonitrile and added dropwise to the bipyridine solution. The mixture was refluxed for 3 days. The precipitate was filtered off, washed with acetonitrile and dried in vacuo to give compound 5.

(4) Intermediate formationSynthesis of Compound 6: compound 5 and tetrabutylammonium iodide were dissolved in dry acetonitrile and heated at 85 ℃ for 72h. Centrifuging to obtain precipitate, and vacuum drying. By adding excess NH ₄ PF ₆ After anion conversion, a pale yellow solid was obtained.

(5) Synthesis of TPE-cyc: to the acetonitrile solution of compound 6 was added an excess of tetrabutylammonium chloride and stirred overnight at room temperature. The mixture was then centrifuged and washed with acetonitrile. And (5) drying in vacuum to obtain the product.

Compared with the prior art, the invention has the beneficial effects that: since TPE-cyc has a large flat rectangular cavity, it is able to specifically recognize NADPH and form a 1:1 host-guest complex. TPE-cyc is not only easy to be phagocytosed by tumor cells, but also can selectively capture intracellular NADPH, thereby breaking the balance of redox reactions in which NADPH participates and improving the antioxidation capability. At the same time, the tumor cells have a high reduction environment, such as high concentration of GSH, and can make TPE-cyc MV ²⁺ Reduction to MV ^+· PET effect between TPE and MV is blocked, fluorescence of TPE-cyc is recovered, and finally high-resolution fluorescence imaging of tumor cells is realized. The cationic cyclophilin compound opens up a new path in the aspect of biological application of identifying biomacromolecules and imaging tumor cells, and has great potential in the aspect of diagnosing and treating human difficult and complicated diseases in the future.

Description of the drawings:

FIG. 1 is a section ¹ H NMR spectrum (D) ₂ O, room temperature, 400 MHz) NADPH (2.00 mM),

(2.00mM)，TPE-cyc(2.00mM)；

FIG. 2 is cytotoxicity of HeLa and U87 cells incubated with different concentrations of TPE-cyc for 4h and 24 h;

FIG. 3 is a fluorescence image of ROS in HeLa cells incubated for various times with TPE-cyc;

FIG. 4 is a flow cytometry detection of ROS fluorescent signals in HeLa cells incubated for different times;

FIG. 5 is a graph with or without Na ₂ S ₂ O ₄ The ultraviolet visible spectrum of TPE-cyc;

FIG. 6 is a graph with or without Na ₂ S ₂ O ₄ The fluorescence spectrum of TPE-cyc;

FIG. 7 is a confocal image of HeLa cells incubated for various times with TPE-cyc (nuclei of living HeLa cells were stained with Nuclear Green LCS 1).

Detailed Description

The technical scheme of the invention is described below through specific embodiments. It is to be understood that the mention of one or more method steps of the present invention does not exclude the presence of other method steps before and after the combination step or that other method steps may be added between these explicitly mentioned steps; it is also understood that these examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.

The invention is further illustrated by the following examples.

Example 1: synthesis of Compound 3

At 0 ℃, N ₂ 37.1mL of n-butyllithium (1.6M) was added to a solution of compound 1 (10 g,59.4 mmol) in THF (100 mL) under an atmosphere, and the resulting orange-red solution was stirred at 0deg.C for 30min. Then, compound 2 (6.3 g,29.7 mmol) was dissolved in THF (20 mL) to obtain a THF solution of compound 2, then the solution of compound 2 was added dropwise to a mixed solution of compound 1 and n-butyllithium, and the mixed solution was warmed to room temperature and stirred for 6 hours. The reaction was quenched with 60mL of saturated aqueous ammonium chloride. Extraction of the organic phase with DCM with anhydrous Na ₂ SO ₄ And (5) drying. The crude product obtained was dissolved in 20mL of toluene, catalytic amounts of p-toluene sulfonic acid (349mg, 1.8 mmol) were added and the reaction was refluxed at 90℃for 5h, using a solution containing

The molecular sieve dehydration device separates the generated water. By mass fraction of 10% NaHCO ₃ Washing toluene mixture with aqueous solution, and washing toluene mixture with anhydrous Na ₂ SO ₄ After drying, toluene was removed by rotary evaporation, and the crude product was purified by column chromatography (silica gel; petroleum ether as eluent), and the eluent containing the objective compound was collected, dried by spin-drying, and then dried under vacuum at 50℃for 12 hours to give 3 (9.0 g, yield 85%) as a white solid.

Example 2: synthesis of Compound 4

At N ₂ Compound 3 (1.1 g,2.83 mmol) was first dissolved in CCl under an atmosphere ₄ To (20 mL) was then added NBS (1.5 g,8.49 mmol) and dibenzoyl peroxide (50 mg,0.2 mmol), and the mixture was finally heated at 70℃under reflux for 12h. After the reaction, the mixture was filtered to remove solid impurities. Extraction with DCM and extraction of the organic phase with anhydrous Na ₂ SO ₄ And (5) drying. The solvent was removed by rotary evaporation, and the crude product was purified by column chromatography (silica gel; petroleum ether to ethyl acetate volume ratio 10:1 as eluent), and the eluent containing the objective compound was collected, dried by spin, and then dried under vacuum at 50℃for 12 hours to give compound 4 (0.9 g, yield 60%).

Example 3: the synthesis of the compound (5) was carried out,

4,4' -bipyridine (2.0 g,12.8 mmol) was dissolved in 20mL acetonitrile and heated at 75deg.C under reflux. A solution of compound 4 (1.2 mg,2.3 mmol) in acetonitrile (5 mL) was then added dropwise to the bipyridine solution. Reflux is carried out for 3 days at 75 ℃. The mixture after the reaction was filtered, and the cake was washed 3 times with acetonitrile. Vacuum drying at 50℃for 12h gave compound 5 (1.7 g, 90% yield).

Example 4: synthesis of Compound 6

Compound 4 (220 mg,0.42 mmol), 5 (400 mg,0.42 mmol) and tetrabutylammonium iodide (35 mg,0.095 mmol) were dissolved in acetonitrile (100 mL) and reacted at 85℃for 72h. The orange precipitate was obtained by centrifugation and dried under high vacuum. Adding excess NH ₄ PF ₆ After anion conversion (100 mg,1.15 mmol), the precipitate was collected by centrifugation (6000 rpm,5 min) and dried in vacuo to give compound 6 (250 mg, 37% yield) as a pale yellow solid.

Example 5: synthesis of TPE-cyc

To a solution of compound 6 (100 mg,0.074 mmol) in 20mL of acetonitrile was added an excess of TBACl (205.66 mg,0.74 mmol) and stirred overnight at room temperature. Centrifugation (6000 rpm,5 min) gave a precipitate which was washed three times with acetonitrile. After drying in vacuo, the final product TPE-cyc (61 mg, 90% yield) was obtained.

Example 6:

at D ₂ The host-guest interactions between TPE-cyc and NADPH were studied in O. As shown in FIG. 1, 3.3mg of TPE and 1.5mg of NADPH were dissolved in 1mL of deuterated water to prepare a mixed solution of 2mM NADA PH and 2mM PE. The above NADPH and TPE-cyc were used to dissolve in D equimolar ₂ The nuclear magnetic hydrogen spectrum is carried out in the solution prepared by O, and obvious chemical shift change of protons on TPE-cyc is observed. For example, proton H on TPE-cyc ₁ 、H ₂ And H ₃ Is divided into groups of sharp signals and has a pronounced chemical shift change, which may be caused by electrostatic interactions between the tetraphosphoric acid groups of NADPH and the pyridine units. At the same time, H after complexation _4-8 The signal of the proton completely disappeared, and these results all indicate that a host-guest complexation between TPE-cyc and NADPH occurred.

Example 7:

in cytotoxicity experiments, NADPH is considered a key coenzyme in the process of cell electron transfer, driving the biosynthesis of amino acids, DNA, phospholipids, fatty acids and steroids. Another important function of NADPH is its strong reducibility, and the promotion of activities of various enzymes such as glutathione peroxidase (GSHPx), catalase and superoxide dismutase. Thus, breaking the balance of NADPH in the living system can cause serious damage to cells, even death. Considering the strong complexation of NADPH by TPE-cyc, we studied the effect of TPE-cyc on cell biology function. Cell viability was first assessed by 3- (4 ',5' -dimethylthiazol-2 ' -yl) -2, 5-diphenyltetrazolium ammonium bromide (MTT) assay in which U87 and HeLa cells were incubated with varying concentrations of TPE-cyc at 37℃in serum-free DMEM, 5% CO ₂ Incubating in incubator for 4h or 24h. As shown in FIG. 2, the cell viability of 0.063-1. Mu.M was maintained at almost 100%, indicating TPE-cyc does not disrupt NADPH balance in this concentration range. However, with further increases in concentration, survival decreases. When the concentration reached 4. Mu.M, the viability was reduced by half, indicating that this concentration of TPE-cyc could capture sufficient intracellular NADPH and eventually induce cell death.

Example 8:

NADPH has an important ability to keep glutathione reduced to GSH, which with the help of GSHPx eliminates ROS and converts harmful hydrogen peroxide to water. Thus, NADPH plays a vital role in the resistance of cells to oxidative stress. Next, we used 2',7' -dichlorofluorescein diacetate (DCFH-DA) as a fluorescent probe to monitor intracellular ROS levels. DCFH-DA itself does not fluoresce, but ROS are able to oxidize non-fluorescent DCFH to produce fluorescent DCF. As shown in fig. 3, the ROS level of TPE-cyc group decreased with the increase of incubation time, indicating that the antioxidant capacity of cells was decreased. Flow Cytometry (FCM) is also used to quantitatively analyze intracellular ROS levels. The flow results shown in fig. 4 are similar to those of fluorescence imaging, with significantly reduced active oxygen production over time. The reason for this may be that the capture of intracellular NADPH by TPE-cyc breaks the balance of NADPH in the redox reaction, promoting the production of reduced species and thus reducing ROS content.

Example 9:

due to the PET effect between TPE and bipyridine units, water-soluble TPE-cyc is a quenching type host with strong uv absorption without fluorescence, severely limiting its application. Under reducing conditions, MV ²⁺ Can be reduced to MV ^+· (bipyridyl groups) thereby blocking the PET effect between TPE and bipyridyl units, which can be used to excite the fluorescence of TPE-cyc. As shown in FIG. 5, na is added ₂ S ₂ O ₄ After that, MV appeared in the range of 450-750nm ^+· Shows the MV with the help of the reducing agent ²⁺ Is reduced to MV ^+· . As expected, TPE-cyc+Na ₂ S ₂ O ₄ The new fluorescence emission in the 300-550nm range (as shown in FIG. 6) shows that the fluorescent switch of TPE-cyc has been turned on.

Example 10:

the internalization behavior of TPE-cyc was studied using a laser confocal microscope (CLSM). As shown in FIG. 7, after incubation of HeLa with serum-free DMEM containing 2. Mu.M TPE-cyc, a clear blue fluorescence of TPE-cyc was observed at an excitation wavelength of 350nm in the cytoplasm after 2h incubation, indicating that TPE-cyc was easily internalized by HeLa cells. As the incubation time increases to 8h, the blue fluorescence intensity increases significantly, indicating that endocytosis of TPE-cyc is time dependent, and the intracellular reducing environment can ensure continuous luminescence of TPE-cyc, which provides important advantages for application of TPE-cyc in fluorescence imaging.

Claims (8)

1. An application of hydrophilic TPE tetracationic ring shown in formula (I) in preparing medicine for inducing tumor cell death,

2. the use according to claim 1, wherein: the cells are U87 cells or HeLa cells.

3. The application according to claim 1, characterized in that it is: the cells were incubated in cell culture medium containing 4-10. Mu.M of hydrophilic TPE tetracationic ring of formula (I) for 24-48h to induce cell death.

4. A use according to claim 3, wherein: the concentration of the hydrophilic TPE tetracationic ring represented by formula (I) was 4. Mu.M, and the incubation time was 24 hours.

5. An application of a hydrophilic TPE tetracationic ring shown in a formula (I) in preparing a cytoplasm high-resolution fluorescence imaging agent of tumor cells,

6. the use according to claim 5, wherein: the cells are HeLa cells.

7. The application according to claim 5, characterized in that the application is: the cells were incubated in a cell culture medium containing 2-4. Mu.M of hydrophilic TPE tetracationic ring of formula (I) for 2-8h, i.e.blue fluorescence was observed under a high resolution fluorescence imager.

8. The use according to claim 7, wherein: the concentration of the hydrophilic TPE tetracationic ring represented by formula (I) was 2. Mu.M.

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