CN111057009B - Cyanovinylene derivative fluorescent dye and preparation method and application thereof - Google Patents

Abstract

The invention discloses a cyanoethenoid derivative fluorescent dye and a preparation method and application thereof, and discloses a compound shown as a structural general formula (I), wherein the compound R₁Independently selected from H, NH₂，OH，CN，CH₃，COOH，SO₃H, F, Cl, Br or NO₂；R₂Is composed of

. The compound probe molecule can realize the quantitative detection of beta-Gal in a buffer solution test system without being interfered by other protease, biological thiol, active oxygen and common cations, and has been successfully applied to SKOV3 cells to carry out the in-situ detection of beta-Gal in biological cells

Description

Cyanovinylene derivative fluorescent dye and preparation method and application thereof

Technical Field

The invention relates to a cyanoethenoid derivative fluorescence sensor and a preparation method thereof, and the sensor is used for detecting aging-related fluorescence in living cellsβGalactosidase (a), (b), (c), (d) and (d)βGal), belonging to the technical field of chemical sensors.

Background

Cancer is a general term for a large group of malignant tumors. For most cancer patients, early and accurate diagnosis of cancer is critical to improve patient survival and relieve pain. Therefore, clinical analysis and detection of cancer biomarkers are of great significance for the diagnosis, treatment and management of early-onset cancers.βGal as a hydrolase in enzyme-linked immunosorbent assays (ELISA) (Armenta, R.; Tarowski, T.; Gibbons, I.; et al.Anal. Biochem.1985, 146211-; 219), gene expression studies (Nolan, G.P.; engineering, S.; Nicolas, J. -F.; et al.Proc. Natl. Acad. Sci. U. S. A. 1988, 852603-, J.; Magee, J. G.; et al. Zentralbl. Bakteriol. 1994, 280,476-487) and in situ hybridization procedures (Fields, S.; Sternglanz, R.Trends Genet.1994, 10286-.βAbnormalities in Gal activity and concentration are usually closely linked to the onset of aging or ovarian cancer. In excess ofβGal separates the side chains of aminopolysaccharides from core proteins by hydrolysis of glycosidic bonds, leading to disassembly of macromolecular proteoglycans and disruption of basement membrane, extracellular space barriers, thereby promoting infiltration and spreading of cancer cells (Chatterjee, S. K.; Bhattacharya, M.; Barlow, J.).Cancer Res. 1979, 39, 1943-1951). Thus, real-time monitoring in living cellsβThe dynamic distribution and concentration variation of Gal have become an important subject in the fields of cell biological research and clinical diagnosis. Conventional detectionβThere are many methods for Gal activity, such as colorimetry (Gary, R.; Kindell, S).Analytical Biochemistry2005, 343329- & 334), electrochemical methods (chikkaveeraniah, b.; bharde, a.; Morgan, n.; et al.ACS nano2012, 66546-.Bioconjugate Chemistry2008, 19441-βThe real-time in-situ nondestructive detection of the Gal content is urgently needed to develop a more convenient, simple and sensitive detection methodβGal detection method.

In recent years, optical imaging, including ultraviolet, visible and Near Infrared (NIR) light, has become a portable tool for non-invasive visual detection of biological samples with its advantages of high sensitivity, fast response, high spatial and temporal resolution and real-time imaging capability (Li, X.; Gao, X.; Shi, W.; et al).Chem. Rev. 2014, 114, 590-659; Zhang, J. F.; Zhou, Y.; Yoon, J.; et al. Chem. Soc. Rev.2011, 40, 3416-3429). Over the past decade, detectionβGal small molecule fluorescent probes get a lot of positive feedback, due to their small size,low cost, easy chemical modification, wide application range and the like, and can be widely used for cell staining and in vitro detection. In the practical application process, the selection of fluorescent molecules with excellent spectral performance is particularly important for a fluorescence analysis method. However, the defects of photophysical and chemical properties of some fluorescent probe molecules restrict further development of applications. Therefore, the development of novel fluorophores for the identification of tumor markers is an important research direction in the biomedical field at present.

Disclosure of Invention

The invention aims to provide a benzimidazole-cyanoethylene derivative with excimer (eximer) luminescent property, a synthetic method thereof and detection in cellsβ-use of Gal.

The invention is realized as follows:

a compound shown in a structural general formula (I),

wherein R is₁Independently selected from H, NH₂，OH，CN，CH₃，COOH，SO₃H, F, Cl, Br or NO₂。

R₂Is composed of

。

In the above compound (I), R₁The function of the substituent is to adjust the degree of association of the two dyes and thus the spectral properties, R₂The substituent functions to increase the solubility of the dye in physiological aqueous solution and to serve as a responsive group to an analyte. Preferably R₁Is H, R₂Is composed of

。

The preparation method of the compound (I) comprises the following steps:

(A) the compound (1) and the compound (2) are subjected to condensation reaction in alcohol at room temperature under the condition of no catalyst or organic amine as a catalyst to obtain a compound (3),

wherein the organic amine catalyst is selected from piperidine, triethylamine and pyridine; the alcohol is selected from methanol, ethanol, and isopropanol;

(B) refluxing the compound (3) and the compound (4) in an acetonitrile solvent, and using an inorganic base or an organic base as an acid-binding agent to bind generated hydrogen bromide to obtain a compound (5), wherein the feeding ratio of the compound (3) to the compound (4) is 1: 2-1: 5,

wherein the inorganic base is selected from potassium carbonate, sodium carbonate, cesium carbonate or sodium hydroxide; the organic base is selected from triethylamine, diethylamine, N, N-diisopropylethylamine, N-Dimethylformamide (DMF), piperidine or piperazine.

The compound shown in the general formula (I) can be applied to cell fluorescence imaging and can also be applied to cell fluorescence imagingβQuantitative fluorescence detection of Gal.

Benzimidazole-cyanovinylene fluorophore has special excimer (eximer) luminescent property, and as the concentration of a solution increases, the fluorescence of a single molecule is quenched and a new band appears at a long wave, and the special property can compensate the defect of the fluorescence quenching of other dyes at high concentration. The invention synthesizes a novel benzimidazole-cyanovinylene fluorescent dye (I) through the condensation reaction between 2-benzimidazole acetonitrile and 4- (N, N-diethyl) aminobenzaldehyde derivatives, researches the photophysical properties of the dye, and constructs a detection methodβGal fluorescent probes, pairs of such probesβGal has a very sensitive response and was successfully applied to SKOV3 cellsβGal detection and lays a foundation for the development of in vivo imaging.

The invention has the advantages that: (1) The compound has obvious detection effect under high concentration, and makes up the defects of the traditional small molecular fluorescent probe in the detection field. (2) The compounds of the invention can be used to achieve environmental, cellular and in vivo animal performanceβHighly selective, highly sensitive detection of Gal. (3) Due to the fact thatβGal can be specifically recognized and hydrolyzedβGalactose glycosidic linkages, stripping glycosidic groups from the molecular structure, exposing the fluorescent dye matrix, resulting in an increase in the fluorescent signal. Based on the change of the fluorescence signal, the compound probe molecule can realize the effect in a buffer solution test systemβQuantitative detection of Gal without interference from other proteases, biological thiols, active oxygen and common cations. Meanwhile, the probe has been successfully applied to SKOV3 cells for biological intracellular treatmentβ-in situ detection of Gal.

Drawings

FIG. 1 shows the comparison of Compound 12 in different water contents of a Water-acetonitrile Systemβ-fluorescence spectra of Gal detection;

FIG. 2 is a fluorescence spectrum of Compound 12 in different water contents of a water-acetonitrile system;

FIG. 3 is a graph of the water content of compound 12 versus the water content of a water-acetonitrile systemβ-Gal detection of the fluorescence intensity line plot (λ _em= 565 nm)；

FIG. 4 is a graph of the water content of compound 12 versus the water content of a water-acetonitrile systemβ-a uv absorption spectrum of Gal detection;

FIG. 5 shows the pair of compounds 12 in buffer solutions of different pHβ-fluorescence spectra of Gal detection;

FIG. 6 shows the addition of Compound 12 to buffer solutions of varying pHβ-Gal and noneβ-a line plot of fluorescence intensity at 565 nm for the resulting fluorescence spectrum for Gal;

FIG. 7 shows the pair of compounds 12 in buffer solution at pH 4.6β-fluorescence spectra of Gal enzyme concentration gradients;

FIG. 8 shows the pair of compounds 12 in buffer solution at pH 4.6β-uv absorption spectrum of Gal enzyme concentration gradient;

FIG. 9 shows pairs of compounds 12β-selective fluorescence spectrum of Gal;

FIG. 10 is a photograph of a fluorescent image of compound 12 cells;

FIG. 11 is a histogram representation of fluorescence intensity of compound 12 cell fluorescence imaging;

FIG. 12 is a graph showing the results of the cytotoxicity test of Compound 12.

Detailed Description

The experimental methods used in the examples are conventional methods unless otherwise specified, and materials, reagents and the like used therein are commercially available unless otherwise specified.

Example 1

(1) 2-Benzimidazolidacetonitrile (266.0 mg, 1.70 mmol) and 4- (N, N-diethyl) aminobenzaldehyde were weighed out in ethanol (20 mL), followed by dropwise addition of piperidine (1.0 mL, 17.0 mmol) and stirring under inert gas at room temperature for 4 h. After the reaction is finished, a large amount of uniform orange fine sand-shaped precipitates are generated in the solution, the reaction solution is filtered, the mixture is washed by using glacial ethanol, and filter residues are dried to obtain orange powder solid, namely a relatively pure product 3 (396.0 mg, 72%);¹H NMR (400 MHz, DMSO-d₆)：δ = 12.79 (s, 1H), 8.11 (s, 1H), 7.90 (d, 2H, J = 8.4 Hz), 7.63 (d, 1H, J = 7.2 Hz), 7.50 (d, 1H, J = 7.2 Hz), 7.20 (m, 2H, J = 7.2 Hz), 6.84 (d, 2H, J = 8.4 Hz), 3.46 (q, 4H, J = 7.2 Hz), 1.14 (t, 6H, J = 6.4 Hz); ¹³C NMR (100 MHz, DMSO-d₆): δ = 150.6, 149.5, 145.8, 144.0, 135.3, 132.8, 123.2，122.3, 119.6, 119.0, 118.4, 111.8, 111.6, 93.4, 44.4, 12.9; HRMS (ESI): calcd for C₂₀H₂₁N₄ [M+H]⁺ m/z 317.1761, found 317.1806

(2) compound 3 (300.0 mg, 0.95 mmol) and potassium carbonate (131.0 mg, 0.95 mmol) were weighed and triturated and added to acetonitrile (20.0 mL), then benzyl bromide 4 (113. mu.L, 0.95 mmol) was added dropwise, stirred backStream 3 h. After the reaction was completed, the solvent was evaporated under reduced pressure to give a yellow solid, which was purified by column chromatography (eluent: dichloromethane/methanol = 20/1) to give 5 (266.8 mg, 69%) as a yellow solid;¹H NMR (400 MHz, CDCl₃): δ = 7.91 (s, 1H), 7.86 (d, 2H, J = 8.8 Hz), 7.82 (d, 1H, J = 8.0 Hz), 7.26 (m, 6H), 7.13 (d, 2H, J = 6.8 Hz), 6.66 (d, 2H, J = 8.8 Hz), 5.69 (s, 2H), 3.43 (q, 4H, J = 7.2 Hz), 1.21 (t, 6H, J = 7.2 Hz); ¹³C NMR (100MHz, CDCl₃) : δ = 151.7, 150.7, 149.5, 136.5, 136.0, 133.1, 129.0, 128.0, 126.6, 123.5, 123.2, 120.2, 119.6, 118.6, 111.2, 110.4, 48.3, 44.8, 12.7; HRMS (ESI) : calcd for C₂₇H₂₇N₄ [M+H]⁺ m/ z 407.2230, found 407.2272。

EXAMPLE 2 preparation of Compound 12

The compound 3 was prepared in the same manner as in example 1.

(1) Weighing p-hydroxybenzaldehyde 6 (257.0 mg, 2.10 mmol) and dissolving in 1 mol/L sodium hydroxide solution

Wherein 2, 3, 4, 6-tetraacetyl-β-D-glucopyranose 7 (867.0 mg, 2.10 mmol) and the reaction stirred at reflux (60 ± 3 ℃) for 4 h. When the solution was completely yellowish, the solvent was removed by distillation under reduced pressure after the end of the reaction. The obtained solid was subjected to silica gel column chromatography (eluent: ethyl acetate/petroleum ether = 1/3) to obtain compound 8 (598.0 mg, 63%);¹H NMR (400 MHz, CDCl₃) : δ = 9.92 (s, 1H), 7.86 (d, 2H, J = 8.8 Hz), 7.12 (d, 2H, J = 8.0 Hz), 5.51 (d, 2H, J = 17.6 Hz), 5.20 (d, 1H, J= 8.0 Hz), 5.15 (d, 1H, J = 9.6 Hz), 4.16 (m, 3H), 2.195 (s, 3H), 2.07 (s, 6H), 2.026 (s, 3H). HRMS (ESI): calcd for C₂₁H₂₅O₁₁[M+H]⁺ m/z 452.1319, found 452.1667。

(2) dissolving the compound 8 (200.0 mg, 0.44 mmol) prepared in the step (2) in methanol, and then adding hydroboration

Sodium (24.0 mg, 0.88 mmol), reaction checked by TLC. After the reaction is finished, methanol is removed by reduced pressure distillation, then ethyl acetate is used for extraction, distilled water is used for washing, the obtained organic phase is dried by anhydrous sodium sulfate, and then the solvent is removed by reduced pressure distillation. The obtained solid was purified by silica gel column chromatography (eluent: ethyl acetate/petroleum ether = 1/2) to obtain compound 9 (157.8 mg, 79%);¹H NMR (400 MHz, CDCl₃) : δ = 7.30 (d, 2H, J = 8.8 Hz), 6.99 (d, 2H, J = 8.8 Hz), 5.48 (d, 2H, J = 10.4 Hz), 5.13 (d, 1H, J = 8.0 Hz), 5.05 (d, 1H, J = 8.0 Hz), 4.63 (s, 2H), 2.19 (s, 3H), 2.07 (s, 6H), 2.02 (s, 3H). HRMS (ESI): calcd for C₂₁H₂₆NaO₁₁[M+Na]⁺ m/z 477.1367, found 477.1357。

(3) compound 9 (515.0 mg, 1.13 mmol) from step (3) was dissolved in dichloromethane (20.0 mL), phosphorus tribromide (118.4. mu.L, 1.25 mmol) was added under ice bath conditions, and the reaction was stirred for 30 min. After completion of the reaction, the reaction solution was extracted with dichloromethane, washed with cold distilled water, dried with anhydrous sodium sulfate in the organic phase, and then distilled under reduced pressure to remove the solvent. The obtained solid was purified by silica gel column chromatography (eluent: ethyl acetate/petroleum ether = 1/3) to obtain compound 10 (443.1 mg, 76%):¹H NMR (400 MHz, CDCl₃) : δ = 7.34 (d, 2H, J = 8.0 Hz), 6.97 (d, 2H, J = 7.2 Hz), 5.47 (m, 2H), 5.14 (dd, 1H, J = 10.4 Hz), 5.07 (d, 1H, J= 8.0 Hz), 4.49 (s, 2H), 4.21 (m, 1H), 4.19 (m, 1H), 4.11 (m, 1H), 2.19 (s, 3H), 2.04 (s, 6H), 2.02 (s, 3H). HRMS (ESI): calcd for C₂₁H₂₅BrNaO₁₀[M+Na]⁺ m/z539.0523, found 539.0512。

(4) potassium carbonate (131.0 mg, 0.95 mmol) compound 3 (300.0 mg, 0.95 mmol) prepared in step (1) was taken and ground to acetonitrile (20.0 mL), followed by compound 10 (490.0 mg, 0.95 mmol), and the reaction was completed under TLC monitoring. The reaction solution was then extracted with ethyl acetate, washed with distilled water, and the organic phase was dried over anhydrous sodium sulfate and distilled under reduced pressure to remove the organic solvent. The crude product was purified by column chromatography on silica gel (eluent: ethyl acetate/petroleum ether = 1/3) to give 11 (364.3 mg, 51%) as a yellow solid. HRMS (ESI) calcd for C₄₁H₄₄N₄O₁₀ [M+H]⁺ m/z 753.3130, found 753.3110。

(5) Compound 11 (500.0 mg, 0.68 mmol) obtained in step (4) was dissolved in a solution of dichloromethane/methanol = 1/4 (30 mL), and sodium methoxide (3.7 mg, 0.068 mmol) dissolved in 1 mol/L aqueous NaOH was added under ice-water bath conditions, and the reaction was monitored by TLC. After the completion of the reaction, the solvent was distilled off under reduced pressure, and the solid was purified by silica gel column chromatography (dichloromethane/methanol = 30/1) to obtain compound 12 (159.0 mg, 40%); ¹H NMR (400 MHz, CD₃OD) : δ = 7.82 (d, 2H, J = 8.8 Hz), 7.68 (d, 1H, J = 8.8 Hz), 7.63 (s, 1H), 7.44 (d, 1H, J = 8.8 Hz), 7.29 (t, 2H, J = 4.0 Hz), 7.06 (m, 4H), 6.76 (d, 2H, J = 8.8 Hz), 5.64 (s, 2H), 4.81 (s, 1H), 3.86 (d, 1H, J = 3.2 Hz), 3.71 (m, 4H), 3.49 (q, 4H, J = 7.2 Hz), 3.34 (s, 2H), 1.21 (t, 6H, J = 7.2 Hz). ¹³C NMR(100 MHz, CD₃OD) : δ = 158.8, 152.8, 152.4, 150.8, 143.2, 137.3, 134.1, 131.2, 129.5, 128.8, 124.7, 124.3, 120.8, 119.7, 119.3, 118.1, 112.3, 112.0, 102.8, 90.5, 76.9, 74.8, 72.2, 70.1, 62.4, 45.5, 12.9. HRMS (ESI): calcd for C₃₃H₃₇N₄O₆[M+H] ⁺ m/z 585.2708, C₃₃H₃₆N₄NaO₆ [M+Na]⁺ m/z 607.2527, C₃₃H₃₆N₄KO₆ [M+K]⁺ m/z 623.2266, found 585.2709, 607.2539, 623.2265。

example 3

R can be easily prepared by one skilled in the art by a method similar to that of example 2₁Independently selected from NH₂，OH，CN，CH₃，COOH，SO₃H, F, Cl, Br or NO₂，R₂Is composed of

The compound of (1).

EXAMPLE 4 spectroscopic Property testing of Compound 12

Compound 12 with different water content pairs in water-acetonitrile systemβ-effect of Gal fluorescence test: 4 mL of water-acetonitrile mixture (water content) with different proportions was added to a 4 mL centrifuge tubef _w= 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99%), and 10 μ L of probe stock (8 mM in DMSO). Two groups of the same solution were prepared. One group was heated in a 37 ℃ water bath for a while until the temperature of each group reached 37 ℃, at which time it was addedβGal (10U/L), and after reacting for 20 min, the fluorescence spectrum and the ultraviolet absorption spectrum were measured (see FIGS. 1 and 4). The other set was heated in a 37 ℃ water bath for 20 min before direct measurement of the fluorescence spectra (see FIG. 2). For ease of observation, the fluorescence intensity at 565 nm of FIGS. 1 and 2 was extracted to produce a line graph (see FIG. 3). Probe andβafter Gal reactionGet rid ofβThe galactose recognition group moiety, leaving the dye monomer, due to its very poor solubility in aqueous solutions, associates into more stable excimer molecules and red shifts from the originally very weak short wavelength fluorescence to produce intense long wavelength fluorescence. Wherein, the dye monomer has good solubility in a large amount of organic solvent, so that the phenomenon of excimer fluorescence cannot occur;

compound 12 in buffer of different pHβ-fluorescence spectroscopy of Gal: 400. mu.L of 0.2 mM phosphate buffer (PBS, pH 3.7, 4.3, 4.6, 5.1, 5.5, 5.9, 6.3, 6.8, 7.2, 8.0, 9.0, respectively) and 10. mu.L of 8 mM probe stock solution were added to a 4 mL centrifuge tube, and distilled water was added to the mixture to thereby prepare 4 mL of solution, the solution was stirred and heated in a 37 ℃ water bath until the temperature of each solution reached 37 ℃ for a while, and then, the solution was added theretoβGal (10U/L), and after reacting for 20 min, the fluorescence spectra of each group were determined. Due to the fact thatβThe activity interval of Gal is acidic, so when the pH of the test system is gradually changed from basic to acidic, the fluorescence intensity of the system at 565 nm is gradually increased and the optimal emission wavelength is red-shifted from 525 nm to 565 nm (see FIG. 5). To facilitate observation of the trend of the change, a plot of the fluorescence intensity at a wavelength of 565 nm was made for reference (see FIG. 6);

compound 12 pairsβ-Gal concentration working curve determination: from BMZ-Gal pairsβFluorescence spectroscopy of Gal enzyme concentration yields the intensity of the fluorescence response at 565 nm as a function ofβ-Gal enzyme concentration working curve. When the system is added, as shown in FIG. 7βWhen the amount of Gal is gradually increased (0-25U/L), the fluorescence intensity of the system is continuously increased. It can also be seen in the UV absorption spectrum that the absorption at 434 nm is gradually decreasing, while the absorption at 490 nm is constantly increasing (see FIG. 8).

Compound 12 pairsβGal is tested in the selective fluorescence spectra of common cations, proteases, biological thiols and active oxygen: adding 20U/L into each group of solutionβGal, lysozyme, trypsin, xanthine oxidase, glucose and 50. mu. M H₂S，H₂O₂，Hcy，GSH，Cys，DTT，Al³⁺，Fe³⁺，Mg²⁺，Ca²⁺，Cu²⁺. After shaking to constant volume, the fluorescence spectra of each group were measured. As can be seen from FIG. 9, additionβThe fluorescence of the system of-Gal is greatly enhanced at 565 nm, and the addition of other protease, biological thiol and common cation can not cause the change of the fluorescence of the system, thereby indicating that the BMZ-Gal pairβThe detection of Gal is not interfered by other substances, and has excellent selectivity.

Cell imaging study of compound 12: 5% CO at 37 ℃ with 10% fetal bovine serum and 1% penicillin/streptomycin₂ SKOV-3 cells were cultured in DMEM dishes under humidified atmosphere, and the SKOV-3 cells were stained with DAPI as a control. These cells were incubated for 30 min with BMZ-Gal (30U/L) and then D-galactonase (1mM) was added to eliminate the intracellular bulkβGal, addition of hydroxyurea (HU, 20. mu.M) to promote cellular senescenceβ-Gal hypersecretion. A fluorescence imaging picture is taken as shown in fig. 10. The fluorescence intensity values in the figure are plotted as a bar graph for easy observation of the effect (see FIG. 11). The effect of this test system on cytotoxicity was also examined using MTT (see FIG. 12).

Claims (3)

1. A compound represented by the structural formula (I),

。

2. a process for the preparation of a compound according to claim 1, characterized in that it comprises the following steps:

(A) the compound (1) and the compound (2) are condensed in ethanol at room temperature by taking pyridine as a catalyst to obtain a compound (3),

(B) refluxing the compound (3) and the compound (10) in an acetonitrile solvent, and binding generated hydrogen bromide by using sodium carbonate as an acid-binding agent to obtain a compound (11), wherein the feeding molar ratio of the compound (3) to the compound (10) is 1: 2-1: 5,

(C) synthesis of Compound (12) from Compound (11)

。

3. Use of a compound according to claim 1 for the preparation of a compound for cellular fluorescence imaging.

Images