A ready-to-use fluorescent probe for labeling lung cancer target nicotinic acetylcholine receptor and a preparation method thereof

Abstract

The application discloses a no-wash type fluorescent probe for marking a lung cancer target point nicotinic acetylcholine receptor and a preparation method, and specifically relates to the following steps: taking triphenyl bromoethylene and 4-carboxyphenylboronic acid as raw materials, and synthesizing a fluorophore TPE-COOH through a condensation reaction; and performing a reaction on the TPE-COOH and R-3-aminoquinuclidine dihydrochloride to generate a fluorophore TPE-EVP, which is an AIE type fluorescent probe for marking the nicotinic acetylcholine receptor. The method synthesizes the fluorescent probe TPE-EVP for no-wash imaging of nAChRs, can mark the nicotinic acetylcholine receptor, is stable in a water environment under physiological conditions, and can successfully identify 293T cells, A549 cells and nicotine-incubated A549 cells at a cell level.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of analytical detection, and particularly relates to a no-wash fluorescent probe for labeling a lung cancer target point nicotinic acetylcholine receptor, and also relates to a preparation method of the no-wash fluorescent probe. BACKGROUND

[0002] Nicotinic acetylcholine receptors (nAChRs) are a large family of proteins that contain many different members, i.e., different subtypes of proteins. Nicotine intake caused by smoking can affect a series of physiological changes of nicotinic acetylcholine receptors in the body, and plays an important role in many physiological and pathological processes, including cholinergic system dysfunction and some nervous system diseases, such as cancer, inflammation, lung disease and Alzheimer's disease. It can also cause drug addiction and schizophrenia.

[0003] In 2018, it was reported that two fluorescent probes, nifrodansyl and nifrofam, for imaging α4β2-nAChR were designed, synthesized and evaluated. Nifrodansyl and nifrofam showed nanomolar affinity for α4β2-nAChR in [3H]cytisine radiolabeled rat brain sections. Nifrofam labeling was observed in HEK cells expressing α4β2-nAChR and was up-regulated by nicotine exposure. However, as constant fluorescent tags, the two probes cannot detect nAChRs under no-wash conditions, and the detection results are greatly affected by operation. SUMMARY

[0004] The purpose of the present application is to provide a no-wash fluorescent probe for labeling a lung cancer target point nicotinic acetylcholine receptor, which can recognize and label nicotinic acetylcholine receptors at the cellular level.

[0005] The technical solution adopted by the present application is a no-wash fluorescent probe for labeling a lung cancer target point nicotinic acetylcholine receptor, which has the following structural formula as shown in formula (I):

[0006]

[0007] Another technical solution adopted by the present application is a preparation method of a no-wash fluorescent probe for labeling a lung cancer target point nicotinic acetylcholine receptor, which is implemented according to the following steps:

[0008] Step 1: using triphenylbromoethylene and 4-carboxyphenylboronic acid as raw materials, a fluorescent group TPE-COOH is synthesized by condensation reaction;

[0009] Step 2, TPE-COOH and R-3-aminoquinuclidine dihydrochloride are reacted to generate the fluorophore TPE-EVP, which is an AIE-type fluorescent probe for labeling nicotinic acetylcholine receptors.

[0010] The application also has the characteristics that,

[0011] In step 1, specifically, triphenylbromovinyl, 4-carboxyphenylboronic acid, tetrakis(triphenylphosphine)palladium, and potassium carbonate are mixed, and heated at 70-90 DEG C for 8-16 h; then cooled to room temperature, extracted with dichloromethane and brine, and the organic phase is left, and the solvent is removed by vacuum filtration, and the dried solid is purified by column chromatography to obtain the fluorophore TPE-COOH.

[0012] The mass ratio of triphenylbromovinyl, 4-carboxyphenylboronic acid, tetrakis(triphenylphosphine)palladium, and potassium carbonate is 320:215:1380:1.

[0013] In step 2, specifically, dichloromethane, TPE-COOH, R-3-aminoquinuclidine dihydrochloride are mixed, and EDCI and DMAP are added, and stirred at room temperature for 1-3 h, and then separated by column chromatography to obtain the fluorescent probe TPE-EVP.

[0014] The mass ratio of TPE, R-3-aminoquinuclidine dihydrochloride, EDCI, and DMAP is 1.5:1:136:1.6.

[0015] The application has the beneficial effect that the method synthesizes the fluorescent probe TPE-EVP for wash-free imaging of nAChRs, which can label nicotinic acetylcholine receptors, has stable fluorescence intensity in a water environment under physiological conditions, and has good selectivity and is not interfered by other inorganic metal ions and amino acid components; and can successfully identify 293T cells, A549 cells, and nicotine-incubated A549 cells at the cell level. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The principle diagram of the fluorescent probe of the application for detecting nicotinic acetylcholine;

[0017] Figure 2 is the ultraviolet absorbance graph of the probe molecule TPE-EVP under different pH conditions in a water environment;

[0018] Figure 3 is the fluorescence emission intensity graph of the probe molecule TPE-EVP under different pH conditions in a water environment;

[0019] Figure 4 is the fluorescence emission intensity graph of the probe molecule TPE-EVP under different interference conditions in a water environment;

[0020] Figure 5 is the fluorescence emission intensity of the probe molecule TPE-EVP at an emission wavelength of 435 nm at different pH values;

[0021] Figure 6 is a 293T cell staining diagram of the probe molecule TPE-EVP (5 μmol / L);

[0022] Figure 7 is an A549 cell staining diagram of the probe molecule TPE-EVP (5 μmol / L);

[0023] Figure 8 is a nicotine incubated A549 cell staining diagram of the probe molecule TPE-EVP (5 μmol / L). DETAILED DESCRIPTION

[0024] The present application will be described in detail below in conjunction with specific embodiments and drawings.

[0025] The present application is a no-wash fluorescent probe for marking the lung cancer target point nicotinic acetylcholine receptor, and has a structural formula as shown in the following formula (I):

[0026]

[0027] Example 1

[0028] The preparation method of the no-wash fluorescent probe for marking the lung cancer target point nicotinic acetylcholine receptor is specifically implemented according to the following steps:

[0029] Step 1, using triphenyl bromoethylene and 4-carboxyphenylboronic acid as raw materials, a fluorophore TPE-COOH as shown in the following formula (II) is synthesized through a condensation reaction;

[0030]

[0031] Specifically, triphenyl bromoethylene, 4-carboxyphenylboronic acid, tetrakis(triphenylphosphine)palladium, and potassium carbonate are mixed, and heated at 70-90°C for 8-16h; then cooled to room temperature, extracted with dichloromethane and brine, and the organic phase is left, the solvent is removed by vacuum filtration, and the dried solid is purified by column chromatography to obtain the fluorophore TPE-COOH;

[0032] The mass ratio of triphenyl bromoethylene, 4-carboxyphenylboronic acid, tetrakis(triphenylphosphine)palladium, and potassium carbonate is 320:215:1380:1;

[0033] Step 2, TPE-COOH and R-3-aminoquinuclidine dihydrochloride are reacted to generate a fluorophore TPE-EVP, as shown in formula (III);

[0034]

[0035] Specifically, dichloromethane, TPE-COOH, R-3-aminoquinuclidine dihydrochloride are uniformly mixed, EDCI and DMAP are added, and stirring is carried out at room temperature for 1-3 hours, and then column chromatography is carried out to separate and obtain a white powder product, which is the fluorescent probe TPE-EVP.

[0036] The mass ratio of TPE, R-3-aminoquinuclidine dihydrochloride, EDCI and DMAP is 1.5:1:136:1.6.

[0037] The principle of the prepared self-cleaning fluorescent probe for labeling the nicotinic acetylcholine receptor is as shown in the figure. Figure 1 EVP-6124 is a small molecule agonist of the nicotinic acetylcholine receptor, wherein the quinuclidine is protonated and locked in the benzene ring cage of the receptor binding site, the basic bridged amino nitrogen forms a cation-pi interaction, forms a hydrogen bond with the carbonyl oxygen on the Y210 amino acid residue of the binding site, and the carbonyl oxygen forms a hydrogen bond with the amide nitrogen of C212, and the benzothiophene group at the tail extends from the aromatic cage and inserts into the hydrophilic pocket formed by S56, S58, Q79, Q183, D186 and E211. In cell imaging, only weak blue fluorescence can be observed in 293T cells, and bright blue color is presented in A549 cells.

[0038] Example 2

[0039] The preparation method of the self-cleaning fluorescent probe for labeling the nicotinic acetylcholine receptor of lung cancer target points according to the present application is specifically implemented according to the following steps.

[0040] Step 1, triphenylbromoethylene and 4-carboxyphenylboronic acid are used as raw materials to synthesize the fluorescent group TPE-COOH through condensation reaction;

[0041] Specifically, triphenylbromoethylene 668 mg, 4-carboxyphenylboronic acid 432 mg, 2M K2CO3 solution 10 mL, TBAB 24 mg are dissolved in 100 mL round-bottom flask with anhydrous THF 20 mL, and Ar2 is protected under room temperature stirring for 30 minutes. Then, tetra(triphenylphosphine)palladium 20 mg is added, and stirring is carried out at 80 DEG C for 16 hours until the reaction is completed. After the reaction is completed, it is cooled to room temperature, extracted with DCM and saturated brine 100 mL*3 times, the organic phase is reserved, the solvent is removed by reduced pressure distillation, and the crude product is purified by column chromatography.

[0042] Step 2, TPE-COOH and R-3-aminoquinuclidine dihydrochloride are reacted to generate the fluorescent probe TPE-EVP;

[0043] Specifically: TPE-COOH 376 mg, R-3-aminoquinuclidine dihydrochloride 250 mg, EDCI 400 mg, DMAP 400 mg were added into a clean and dry 100 mL round-bottom flask in batches. 20 mL of super-dry dichloromethane was slowly added along the bottle wall. Stir at room temperature at medium speed for 2 hours until the reaction is completely consumed, and the reaction is completed. After the experiment, extract with DCM 30 mL x 3 times and saturated brine 100 mL, collect the organic phase, and evaporate the organic solvent under reduced pressure. Purify by column chromatography to obtain white powder product.

[0044] The product is characterized as follows:

[0045] The structural characterization data of TPE-COOH are as follows: nuclear magnetic resonance characterization: 1 H NMR (400 MHz, DMSO-d6) δ 12.86 (s, 1H), 7.69 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 1.9 Hz, 9H), 7.09 (s, 1H), 7.07 (s, 1H), 6.98 (td, J = 7.2, 1.9 Hz, 6H). 1 HRMS: Calcd for C 27 H 20 O2[M+Na] + : 399.1756; Found: 399.1737.

[0046] The structural characterization data of TPE-EVP are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.21 (d, J = 8 Hz, 1H), 7.93 (d, J = 8 Hz, 2H), 7.45 (dt, J = 8 Hz, 8H), 7.38 (m, 9H), 4.12 (dddd, J = 4 Hz, 1H), 2.97 (dd, J = 4 Hz, 1H), 2.82 (t, J = 8 Hz, 1H), 2.80 (td, J = 8 Hz, 1H), 2.73 (m, 2H), 2.66 (ddd, J = 4 Hz, 1H), 2.10 (q, J = 8 Hz, 1H), 1.70 (m, 4H). HRMS: Calcd for C 34 H 32 N2O[M+H] + : 485.2587; Found: 485.2583.

[0047] Probe pH stability test

[0048] In order to study the stability of the probe TPE-EVP under different pH conditions, such as Figures 2-4As shown, the probe TPE-EVP was selected to test the UV absorption and fluorescence emission intensity signal diagram at different pH under the condition of water as solvent, it was found that the probe TPE-EVP had good pH stability, only when the pH was more than 9, the UV and fluorescence signal changed obviously. In the physiological condition pH = 7, the fluorescence emission wavelength was 493 nm.

[0049] Figure 2 is the UV absorption of the probe TPE-EVP at different pH when the concentration of the probe is 10 μM, it can be found that the absorbance is maximum at 232 nm. Figure 3 is the fluorescence emission wavelength of the probe TPE-EVP at different pH at the maximum UV absorbance 232 nm when the concentration of the probe is 10 μM, the maximum emission reaches 435 nm. Figure 4 is the fluorescence emission intensity of the probe TPE-EVP at different pH at the maximum emission wavelength 432 nm, the emission intensity is maximum when pH = 7, which shows that the probe is suitable for detection under physiological conditions.

[0050] Figure 5 is the fluorescence emission intensity of the probe TPE-EVP at 432 nm at pH = 7 in the presence of a variety of complex interferents. It can be seen that the fluorescence emission intensity can remain stable in the presence of interferents, which shows that the probe is suitable for imaging in complex biological systems.

[0051] Figure 6 is the cell staining image of the probe TPE-EVP for 293T cell washing-free imaging. It can be seen that there is a faint blue color in the picture, which shows that the expression amount of nAChRs in 293T cells is low. Figure 7 is the cell staining image of the probe TPE-EVP for A549 cell washing-free imaging. It can be seen that there is a relatively bright blue color in the picture, which shows that the expression amount of nAChRs in A549 cells is more than that in 293T cells. Figure 8 is the cell staining image of the probe TPE-EVP for A549 cell washing-free imaging after nicotine incubation. It can be seen that there is a very bright blue color in the picture, which shows that the expression amount of nAChRs in nicotine incubated A549 cells is sharply increased.

[0052] Example 3

[0053] The preparation method of the washing-free fluorescent probe for marking the lung cancer target point nAChR is specifically implemented according to the following steps:

[0054] Step 1, mixing triphenylbromovinyl, 4-carboxyphenylboronic acid, tetrakis(triphenylphosphine)palladium, and potassium carbonate, heating at 90 °C for 10 h; then cooling to room temperature, extracting with dichloromethane and brine, leaving the organic phase, removing the solvent by vacuum filtration, and purifying the dried solid by column chromatography to obtain the fluorophore TPE-COOH;

[0055] Step 2, mixing dichloromethane, TPE-COOH, R-3-aminoquinuclidine dihydrochloride, adding EDCI and DMAP, stirring at room temperature for 2 h, and then separating by column chromatography to obtain the white powdery product, i.e. the fluorescent probe TPE-EVP.

Claims

1. A ready-to-use fluorescent probe for labeling a lung cancer target, nicotinic acetylcholine receptor, characterized by, The fluorescent probe has a structural formula shown in the following formula (I): （Ⅰ）。 2. The method for preparing a ready-to-use fluorescent probe for labeling a lung cancer target, nicotinic acetylcholine receptor according to claim 1, wherein the nicotinic acetylcholine receptor is selected from the group consisting of alpha 3, alpha 4, alpha 7, and alpha 9. The following steps are specifically implemented: Step 1, using triphenyl bromoethylene and 4-carboxyphenylboronic acid as raw materials, a fluorophore TPE-COOH is synthesized through a condensation reaction; specifically, triphenyl bromoethylene, 4-carboxyphenylboronic acid, tetrakis(triphenylphosphine)palladium, and potassium carbonate are mixed, and heated at 70-90 DEG C for 8-16 h; then cooled to room temperature, extracted with dichloromethane and brine, the organic phase is left, the solvent is removed by vacuum filtration, and the dried solid is purified by column chromatography to obtain the fluorophore TPE-COOH; Step 2, TPE-COOH and R-3-aminoquinuclidine dihydrochloride are reacted to generate a fluorophore TPE-EVP, which is an AIE-type fluorescent probe for labeling nicotinic acetylcholine receptors; specifically, dichloromethane, TPE-COOH, and R-3-aminoquinuclidine dihydrochloride are mixed uniformly, EDCI and DMAP are added, and stirred at room temperature for 1-3 h, and then separated by column chromatography to obtain the fluorescent probe TPE-EVP.

3. The method for preparing a wash-free fluorescent probe for labeling the nicotinic acetylcholine receptor, a target of lung cancer, according to claim 2, wherein: The mass ratio of triphenyl bromoethylene, 4-carboxyphenylboronic acid, tetrakis(triphenylphosphine)palladium, and potassium carbonate is 320:215:1380:

1.

4. The method for preparing a wash-free fluorescent probe for labeling the nicotinic acetylcholine receptor, a target of lung cancer, according to claim 2, wherein: The mass ratio of TPE, R-3-aminoquinuclidine dihydrochloride, EDCI, and DMAP is 1.5:1:136:1.6.

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