CN110272731B

CN110272731B - Fluorescent probe DCCO and preparation method and application thereof

Info

Publication number: CN110272731B
Application number: CN201910634808.8A
Authority: CN
Inventors: 钞建宾; 段月香; 张永斌
Original assignee: Shanxi University
Current assignee: Shanxi University
Priority date: 2019-07-15
Filing date: 2019-07-15
Publication date: 2021-05-14
Anticipated expiration: 2039-07-15
Also published as: CN110272731A

Abstract

The invention provides a fluorescent probe DCCO and a preparation method and application thereof. The fluorescent probe is 7- (diethylamino) -3- (E) -3- (9-ethyl-9H-carbazole-3-yl) acryloyl) -2H-pyran-2-one. The preparation method comprises the following steps: dissolving 3-acetyl-7- (diethylamino) -2H-pyran-2-one and 9-ethyl-9H-carbazole-3-formaldehyde in an ethanol/acetonitrile (1/1, V/V) mixed solution according to an equal molar ratio, dripping piperidine, heating and refluxing, and changing the solution from orange to red; removing the solvent under reduced pressure, and performing column chromatography separation by a gradient elution method to obtain a crude product of the fluorescent probe; dissolving with diethyl ether, filtering, and drying to obtain pure product. The fluorescent probe pair ClO^‑The detection is ratio-type, shows high sensitivity and good selectivity and stability, and has the advantages of simple and rapid detection process, accurate detection result and the like. The fluorescent probe DCCO can be used for biological sample ClO^‑The method is applied to detection.

Description

Fluorescent probe DCCO and preparation method and application thereof

Technical Field

The invention relates to an organic small molecule fluorescent probe, in particular to a fluorescent probe DCCO and a preparation method thereof, and application of the fluorescent probe to ClO in a biological sample^-Detection of (3).

Background

For over 100 years, ClO^-Is used as a clinical general disinfectant as an effective drug against various microorganisms. In animals and humans, ClO^-Is also considered to be a key bactericide during the immune response, corresponding to the digestion process of neutrophils phagocytizing bacteria. However, in inflammatory states, excessive production of HClO may trigger or exacerbate a variety of diseases, including Alzheimer's disease, cardiovascular disease, atherosclerosis, inflammatory bowel disease, myocardial infarction, organ transplant rejection, and even cancer. Therefore, there is an urgent need to develop a method for detecting ClO in an organism^-A fast and efficient tool.

Methods for detecting hypochlorous acid or hypochlorite exist at present, such as chromatography, chemiluminescence, electroanalysis and the like. However, these methods have low sensitivity and complicated operation, and more importantly, cannot be applied to HClO/ClO in vivo^-Detection of (3). And the fluorescent probe is used as the most powerful tool for detecting the analyteOne of them, because they have high sensitivity, simple operation, do not need to help the complicated instrument to realize the analyte detection, and also have easy visualization intracellular dynamics and high resolution to locate the interested biological molecules, ratio type fluorescent probe has two independent fluorescent signals, can avoid the background interference better. Fluorescence imaging is carried out by a laser confocal microscope, so that the hypochlorite in the organism can be detected. Therefore, there is an urgent need for the synthesis of ClO probes for detecting biological samples, which have the advantages of simplicity, high sensitivity, good selectivity, low detection limit and good photostability^-。

Disclosure of Invention

One of the purposes of the invention is to provide a fluorescent probe DCCO. The other purpose is to provide a preparation method of the probe DCCO, which has simple preparation process and low cost. The third purpose is to provide the application of the probe, namely the application of the probe in ClO in biological samples^-Detection of (3). The probe has the advantages of quick response, high sensitivity, good selectivity and light stability, capability of effectively reducing the interference of the fluorescence of the biological sample and the like.

The invention provides a fluorescent probe DCCO, which is 7- (diethylamino) -3- (E) -3- (9-ethyl-9H-carbazole-3-yl) acryloyl) -2H-pyran-2-ketone, and has a structural formula as follows:

the invention provides a preparation method of a fluorescent probe DCCO, which comprises the following steps:

(1) dissolving 3-acetyl-7- (diethylamino) -2H-pyran-2-one and 9-ethyl-9H-carbazole-3-formaldehyde in an equal molar ratio in an ethanol/acetonitrile (1/1, V/V) mixed solution, adding piperidine dropwise, heating and refluxing for 30 hours until the solution turns red from orange, and tracking the reaction by TLC (V)_{Ethyl acetate}：V_{Petroleum ether}＝1:3)；

(2) After the reaction is finished, removing the solvent under reduced pressure, performing column chromatography separation by a gradient elution method, eluting with ethyl acetate/petroleum ether (1/3, V/V), and then eluting with ethyl acetate/petroleum ether (1/2, V/V) to obtain a crude product of the fluorescent probe;

(3) dissolving the crude product with diethyl ether, filtering and drying to obtain an orange flocculent pure product DCCO.

The synthetic route is as follows:

the invention provides a method for quantitatively detecting ClO by fluorescence^-The method comprises the following steps:

(1) preparing 1mM fluorescent probe DCCO stock solution by using DMSO;

(2) 2.0mL of water/DMSO (1/1, v/v) system and 10.0. mu.L of stock fluorescent probe were added to a fluorescence cuvette and titration experiments were performed on a fluorescence spectrophotometer with ClO^-The fluorescence intensity at 420nm is gradually enhanced, and the fluorescence intensity at 570nm is gradually weakened;

(3) with ClO^-Concentration is plotted on the abscissa as the relative fluorescence intensity I_420nm/I_570nmDrawing a graph for the ordinate and carrying out Sigmoidal fitting, wherein the linear regression equation is as follows: i is_420nm/I_570nm＝0.033*[NaClO]-1.586, NaClO concentration in 10 units^-6mol/L; linear correlation coefficient of R²The optimal linear response range is 53.6 μ M-375.2 μ M, 0.994.

Stability experiments prove that the fluorescent probe is directed to ClO^-The assay of (2) has good light stability.

Experiments prove that common anions and biological mercaptan do not interfere ClO of a system^-The measurement of (1).

The fluorescent probe DCCO of the invention proves that the fluorescent probe DCCO can be used for detecting ClO in biological samples by the laser confocal microscope imaging technology^-And (4) changing.

Compared with the existing fluorescent probe, the synthesized fluorescent probe DCCO has the following advantages: 1. the fluorescent probe of the invention has simple synthesis steps and low cost. 2. The detection method is simple and can be realized only by means of a fluorescence spectrometer. 3. Fluorescent probeDCCO to ClO^-The response has the characteristics of short response time, high sensitivity, good selectivity and the like, and is not interfered by common anions and biological mercaptan. 4. The ratio type fluorescent probe effectively eliminates the interference of environmental factors. 5. The fluorescent probe DCCO can be used for ClO in cells, zebra fish and arabidopsis thaliana^-Detection of (3).

Drawings

FIG. 1 example 2 fluorescent Probe DCCO with ClO^-Modified ultraviolet absorption spectrum

FIG. 2 example 3 fluorescent Probe DCCO with ClO^-Varied fluorescence titration map

FIG. 3 example 3 fluorescent probes DCCO vs ClO^-Operating curve of response

FIG. 4 example 4 fluorescent Probe DCCO response to common anions

FIG. 5 example 5Hella cytogram

Figure 6 imaging of zebra fish of embodiment 6

FIG. 7 imaging diagram of Arabidopsis thaliana in example 7

Detailed Description

EXAMPLE 1 preparation of fluorescent Probe DCCO

(1) Dissolving 3-acetyl-7- (diethylamino) -2H-pyran-2-one (0.777g, 3mmol) and 9-ethyl-9H-carbazole-3-carbaldehyde (0.669g, 3mmol) in 15mL of an ethanol/acetonitrile (1/1, V/V) mixed solution, adding piperidine (0.75mL, 7.5mmol) dropwise, refluxing under heating for 30 hours until the reaction solution turns red from orange, and performing TLC (V follow-up reaction)_{Ethyl acetate}：V_{Petroleum ether}＝1：3)；

(2) After the reaction is finished, removing the solvent under reduced pressure, performing column chromatography separation by a gradient elution method, eluting with ethyl acetate/petroleum ether (1/3, V/V) at first, and then eluting with ethyl acetate/petroleum ether (1/2, V/V) to obtain 0.699g of a crude product;

(3) the crude product was dissolved in ether, filtered and dried to give 0.349g of pure product as an orange yellow product with a yield of 25%.

For fluorescent probes¹H NMR characterization, results are as follows:

¹H NMR(600MHz,DMSO-d₆,δ/ppm):δ8.61(s,1H),8.55(s,1H),8.24(d,J＝7.6Hz,1H),7.99(d,J＝15.6Hz,1H),7.91(d,J＝15.6Hz,1H),7.87(d,J＝8.4Hz,1H),7.71(s,1H),7.69(s,1H),7.66(d,J＝8.1Hz,1H),7.50(t,J＝7.6Hz,1H),7.26(t,J＝7.3Hz,1H),6.82(d,J＝8.9Hz,1H),6.63(s,1H),4.48(d,J＝6.8Hz,2H),3.51(d,J＝6.6Hz,4H),1.34(t,J＝6.7Hz,3H),1.16(t,J＝6.6Hz,6H).

¹³C NMR(150MHz,DMSO-d₆,δ/ppm):185.89,160.45,158.60,153.30,148.57,144.41,141.42,140.54,132.70,126.81,126.60,126.31,123.14,122.63,122.47,122.34,121.19,120.01,116.52,110.61,110.26,110.08,108.38,96.38,44.91,37.68,14.25,12.85.

EXAMPLE 2 fluorescent Probe DCCO with ClO^-Modified ultraviolet absorption spectrum

ClO was performed by adding 10.0. mu.L of stock fluorescent probe to 2.0mL of water/DMSO (1/1, v/v) system^-Uv titration experiments were performed and uv absorption spectra were recorded (figure 1). With ClO^-As the amount increased, the UV absorption at 290nm, 321nm and 355nm increased, and the UV absorption at 480nm decreased.

EXAMPLE 3 fluorescent Probe DCCO with ClO^-Varied fluorescence titration map

ClO was performed by adding 10.0. mu.L of stock solution of fluorescent probe to a 2.0mL system of water/DMSO (1/1, v/v)^-Fluorescence titration experiment, detection on a fluorescence spectrophotometer, with ClO^-The increase in fluorescence intensity at 570nm gradually decreased, and a new peak appeared at 420nm and gradually increased (FIG. 2). The instrument parameters are that the slit widths of the excitation wavelength and the emission wavelength are respectively 5.0nm and 5.0nm, the voltage is 600V, and the maximum excitation wavelength of the fluorescent probe solution is as follows: lambda [ alpha ]_exAt 335nm and a maximum emission wavelength of lambda_em570 nm. By the ratio of fluorescence I_420nm/I_570nmPlotting the plot for ordinate to obtain ClO^-Working curve of concentration, linear regression equation is I_420nm/I_570nm＝0.033*[NaClO]-1.586, NaClO concentration in 10 units^-6mol/L; linear correlation coefficient of R²The optimal linear response range was 53.6 μ M-375.2 μ M (fig. 3), 0.994.

Example 4 response of fluorescent probes to common anionic and Biothiols

Add 10.0. mu.L of fluorescent probe stock to a 2.0mL system of water/DMSO (1/1, v/v), and add additional anion and biological thiol (F) respectively^-、Ac^-、CNS^-、S^2-、SO₃ ^2-、CO₃ ^2-、Br^-、HCO₃ ^-、SO₄ ^2-、S₂O₃ ^2-、Cl^-、HSO₃ ^-GSH and Cys) to the final concentration of 250 μ M, respectively measuring the fluorescence spectra, and plotting the corresponding fluorescence intensity ratios I of different anions and biological thiol_420nm/I_570nmIs shown in the figure. Experiments prove that other anions and biological mercaptan do not interfere the ClO pair system^-Detection of (2) (fig. 4).

Example 5 imaging of Hella cells

HeLa cells in DMEM medium, 5% CO₂And culturing at 37 ℃. Prior to CLSM imaging, cells were seeded on 14mm glass coverslips and incubated for 12 hours, and the cells were plated out into multiple small petri dishes prior to use. The culture solution of the two small petri dishes was extracted and washed with PBS buffer solution at pH 7.40, and then, 2.0ml PBS (pH 7.40) buffer solution containing 20.0 μ L of probe (1mM, dissolved in DMSO) was added to each of the two small petri dishes to incubate for about 5min, followed by extraction and three-time washing with PBS buffer solution at pH 7.40. To one of the small petri dishes was added a solution containing 10.0. mu.L (1X 10)^-2M)ClO^-The cells were incubated in 2.0mL of a PBS buffer solution with pH 7.4 for about 10min, and then extracted, followed by washing with a PBS buffer solution with pH 7.40. Finally, the cells incubated with the probe and the hypochlorite are respectively placed in a small culture dish, and 2.0mL of PBS solution with pH value of 7.40 is added to observe under a laser confocal microscope. The fixed excitation wavelength is 405nm, and the collection emission bands are a blue channel (405-. As can be seen from FIG. 5, when only the probe was added, the cells exhibited strong yellow fluorescence and weak blue fluorescence (A1-D1), and HSO was added₃ ^-The latter yellow color decreased, while the blue fluorescence increased (A2-D2).

Example 6 zebra fish imaging

Zebra fish cultured in E3 medium and maintained at 28 deg.C, and 5-day-old zebra fish incubated in culture solution containing 10 μ M probe for 15min, and part of the zebra fish was used for imaging, and the other part was incubated in culture solution containing ClO^-The culture broth (μm) was further incubated for 10min, and zebrafish were rinsed three times with PBS buffer before imaging. The fixed excitation wavelength is 405nm, and the collection emission bands are a blue channel (405-. As shown in FIG. 6, after incubation with the probe, zebrafish fluoresced strongly in the yellow channel and weakly in the blue channel (A1-D1). When further ClO is used^-After incubation, the yellow fluorescence decreased and the blue fluorescence increased (A2-D2).

EXAMPLE 7 Arabidopsis thaliana imaging

The method comprises the following steps of (1) disinfecting arabidopsis seeds, sowing the seeds on an MS/2 solid culture medium, carefully and freehand transferring the seeds to a self-made float after 10 days, and putting the seeds into a culture solution; after two days, the seedlings were transferred to a perforated foam plate (the holes were slightly smaller than the floats) and the seedlings after 10 days were taken as experimental materials for future use. Before imaging experiment, using tweezers to take small segments of fibers of arabidopsis thaliana, washing the fibers with PBS buffer solution, putting the fibers into culture solution containing 10 mu M fluorescent probe, incubating for 15min, washing and imaging a part of the fibers, adding ClO into the other part of the fibers^-Culture broth (μm), further incubation for 10min, washing and imaging. The fixed excitation wavelength is 405nm, and the collection emission bands are a blue channel (405-. As shown in FIG. 7, after incubation with the probe, Arabidopsis fluoresced strongly in the yellow channel and weakly in the blue channel (A1-D1). When further ClO is used^-After incubation, the yellow fluorescence decreased and the blue fluorescence increased (A2-D2).

Claims

1. A fluorescent probe DCCO is characterized in that the structural formula is as follows:

2. the method for preparing fluorescent probe DCCO according to claim 1, wherein the synthetic route is as follows:

3. the method for preparing the fluorescent probe DCCO according to claim 1, comprising the steps of:

(1) dissolving 3-acetyl-7- (diethylamino) -2H-pyran-2-one and 9-ethyl-9H-carbazole-3-formaldehyde in an equal molar ratio in a mixed solution of ethanol and acetonitrile with the same volume, dripping piperidine, heating and refluxing for 30 hours until the solution turns red from orange, and tracking the reaction by TLC;

(2) after the reaction is finished, removing the solvent under reduced pressure, and performing column chromatography separation by using a gradient elution method, wherein the volume ratio of the solvent to the solvent is 1: 3, eluting with ethyl acetate and petroleum ether, and then adding a solvent with a volume ratio of 1: 2, eluting the eluent of ethyl acetate and petroleum ether to obtain a crude product of the fluorescent probe;

(3) dissolving the crude product with diethyl ether, filtering and drying to obtain an orange pure product DCCO.

4. Quantitative fluorescence detection ClO^-The method is characterized by comprising the following steps:

(1) preparing 1mM stock solution of the fluorescent probe DCCO according to claim 1 in DMSO;

(2) mixing 2.0mL of the mixture in a volume ratio of 1: 1 System of Water and DMSO and 10.0. mu.L of DCCO stock solution were added to a fluorescence cuvette and the titration experiment was performed on a fluorescence spectrophotometer with ClO^-The fluorescence intensity at 420nm is gradually enhanced, and the fluorescence intensity at 570nm is gradually weakened;

(3) with ClO^-Concentration is plotted on the abscissa as the relative fluorescence intensity I_420nm/I_570nmDrawing a graph for the ordinate and carrying out Sigmoidal fitting, wherein the linear regression equation is as follows: i is_420nm/I_570nm＝0.033*[NaClO]-1.586, NaClO concentration in 10 units^-6mol/L; linear correlation coefficient of R²0.994, the optimal linear response range is 53.6 μ M-375.2 μ M;

the method is non-disease diagnostic or therapeutic.

5. The fluorescent probe DCCO as claimed in claim 1 for use in ClO of biological sample^-The assay of (1), said assay being non-disease diagnostic or therapeutic.