CN110818703B - Pyrrole-part cyanine derivative fluorescent probe and preparation method and application thereof - Google Patents

Abstract

The invention providesA pyrrole-part cyanine derivative fluorescent probe and a preparation method and application thereof, wherein the chemical structural formula of the pyrrole-part cyanine derivative is as follows:

(ii) a The preparation method comprises the following steps: dissolving N-morpholinoethyl-2, 4-dimethyl-5-formylpyrrole-3-formamide and N-ethylbenzothiazole iodide in an organic solvent; dripping piperidine into the obtained solution to be used as a catalyst, and then refluxing and stirring the solution at the temperature of 80 ℃ to react for 3 to 4 hours; and then cooling to room temperature, carrying out vacuum filtration, washing the obtained solid residue with ethanol, and recrystallizing with ethanol to obtain the pyrrole-part cyanine derivative fluorescent probe. The pyrrole-part cyanine derivative fluorescent probe can selectively react with hypochlorite under the condition of pure water phase physiology, the solution is yellow and faded, and meanwhile, green fluorescence is obviously weakened, and the pyrrole-part cyanine derivative fluorescent probe is particularly used as a fluorescent probe for conveniently detecting the hypochlorite in a cell lysosome.

Description

A kind of pyrrole-merocyanine derivative fluorescent probe and its preparation method and application

technical field

The invention belongs to the field of organic synthesis, in particular to a pyrrole-merocyanine derivative and a preparation method and application thereof.

Background technique

Hypochlorous acid (HClO) usually exists in the physiological environment in the form of hypochlorite (ClO−), which has a bactericidal effect and also plays an immune role when the body is invaded by microorganisms. However, excess HClO produced in phagocytes can also be harmful to the human body. There is already evidence that hypochlorite is involved in inflammation of some tissues, and hypochlorite released by neutrophils is thought to be involved in lung damage, rheumatoid arthritis, liver ischemia-reperfusion injury, and kidney disease. Intracellular excess hypochlorite is mainly metabolized by lysosomes, and its concentration is closely related to lysosomal redox balance. In daily life, hypochlorous acid is also a commonly used disinfectant. Therefore, the development of selective and sensitive tools for the detection of hypochlorite in biological samples is increasingly important.

In recent years, fluorescent molecular probe technology has become an important means to detect important metal ions, anions and small molecules due to its high sensitivity, simple operation and low cost. However, most of the existing hypochlorite fluorescent probes require organic co-solvents (>10%), which cannot realize hypochlorite recognition in pure water, which limits their further practical applications. Moreover, there are not many reports of hypochlorite fluorescent probes targeting lysosomes.

In view of this, the present invention is proposed.

SUMMARY OF THE INVENTION

In view of the problems existing in the prior art, the present invention takes into account the excellent photochemical and photophysical properties of merocyanine derivatives, uses pyrrole-containing merocyanine derivatives as fluorescent probes, and introduces morpholine rings as lysosome localization A highly sensitive and selective hypochlorite fluorescent probe was synthesized. The probe can be applied to the determination of hypochlorite in pure water system, has lysosome targeting function, and can be applied to the detection of hypochlorite concentration in lysosomes.

The main purpose of the present invention is to provide a pyrrole-merocyanine derivative fluorescent probe with high sensitivity and good selectivity for hypochlorite in pure water systems and cell lysosomes; another purpose is to provide the fluorescent probe Needle preparation method and application.

In order to achieve the above object, the present invention adopts the following technical scheme: a fluorescent probe of a pyrrole-merocyanine derivative, and the pyrrole-merocyanine derivative has the following structural formula:

。

.

The present invention also provides a preparation method of a pyrrole-merocyanine derivative fluorescent probe, and the specific preparation method is as follows:

S1: N-morpholinoethyl-2,4-dimethyl-5-formylpyrrole-3-carboxamide and N-ethylbenzothiazole iodonium salt are first dissolved in an organic solvent;

S2: Add piperidine dropwise to the solution obtained from S1 as a catalyst, and reflux at 80°C for 3-4h;

S3: the solution obtained in S2 is cooled to room temperature, filtered under reduced pressure, the obtained solid residue is washed with ethanol, and then recrystallized with ethanol to obtain the pyrrole-merocyanine derivative fluorescent probe.

Further, the ethanol is anhydrous ethanol.

Further, the time of the reflux stirring reaction in step S2 is 3h.

Further, in step S2, the molar ratio of N-morpholinoethyl-2,4-dimethyl-5-formylpyrrole-3-carboxamide and piperidine is 1:0.02.

Further, the mol ratio of N-morpholinoethyl-2,4-dimethyl-5-formylpyrrole-3-formamide and N-ethylbenzothiazole iodonium salt added in step S1 is 1: 1.

Further, the specific preparation method is as follows: 2.79 g, N-morpholinoethyl-2,4-dimethyl-5-formylpyrrole-3-carboxamide (10 mmol) and 3.05 g N-ethylbenzene The thiazole iodonium salt (10 mmol) was dissolved in 0.05 L of ethanol, 0.017 g of piperidine (0.2 mmol) was added dropwise as a catalyst, refluxed and stirred at 80 °C for 3-4 h, cooled and allowed to stand to room temperature, and filtered under reduced pressure. Wash with ethanol to obtain the pyrrole-merocyanine derivative fluorescent probe.

The present invention also provides a use of the above-mentioned fluorescent probe of pyrrole-merocyanine derivatives, that is, the application as a hypochlorite fluorescent probe, especially as a detection method for hypochlorite in lysosomes of HeLa living cells. Applications of fluorescent probes.

Compared with the prior art, the advantages and positive effects of the present invention are:

The invention prepares the pyrrole-merocyanine derivative fluorescent probe through condensation reaction, the raw materials are easily available, and the synthesis and post-processing methods are simple. Among a variety of common anions and reactive oxygen species, hypochlorite showed high fluorescence recognition performance. The working environment of the probe does not require any organic solvent to assist solubilization, which is very beneficial to be applied to biological systems and has a wide range of potential application values.

Description of drawings

Fig. 1 is the ¹ H NMR spectrum of the pyrrole-merocyanine derivative fluorescent probe prepared in Example 1 of the present invention;

Figure 2 is the ¹³ C NMR spectrum of the pyrrole-merocyanine derivative fluorescent probe prepared in Example 1 of the present invention;

Fig. 3 is the mass spectrogram of the pyrrole-merocyanine derivative fluorescent probe prepared in Example 1 of the present invention;

Figure 4 is a citric acid-sodium citrate buffer solution (0.02 mol/L, pH = 4) of the pyrrole-merocyanine derivative fluorescent probe (1×10 ^-5 mol/L) prepared in Example 1 of the present invention Add 1×10 ^-4 mol/L anions (AcO ⁻ , Br ⁻ , Cl ⁻ , ClO ⁻ , ClO ₄ ⁻ , CN ⁻ , F ⁻ , H ₂ PO ₄ ⁻ , HPO ₄ ⁻ , I ⁻ , PO ₄ ^{3 −} , S ²⁻ and SO ₃ ²⁻ ) or reactive oxygen species (H ₂ O ₂ , ¹ O ₂ , ·OH, NO and O ₂ ⁻ ) UV (a) and fluorescence (b) spectra (excitation wavelengths of 465 nm);

Figure 5 is a citric acid-sodium citrate buffer solution (0.02 mol/L, pH = 4) of the pyrrole-merocyanine derivative fluorescent probe (1×10 ^-5 mol/L) prepared in Example 1 of the present invention Ultraviolet (a) and fluorescence (b) spectra of titrated ClO ⁻ with different concentrations, the inset shows the linear change trend of the absorbance at 465 nm and the fluorescence intensity at 520 nm with the concentration of hypochlorite (excitation wavelength is 465 nm);

Figure 6 shows the fluorescence imaging of pyrrole-merocyanine derivative fluorescent probe and ClO ⁻ in HeLa cells; HeLa cells were incubated with 1×10 ^-5 mol/L fluorescent probe for 30 minutes and then added 1×10 ^-4 mol/L ClO ⁻ , and after incubation for 30 minutes, fluorescence imaging was performed using an Olympus FV500-IX70 laser confocal microscope.

Among them: a is the green channel fluorescence imaging image of the above-mentioned fluorescent probe; b is the bright-field image of the above-mentioned fluorescent probe; c is the superimposed picture of the above-mentioned fluorescent probe bright-field image and the fluorescent image; d is the above-mentioned fluorescent probe + ClO ⁻ Green channel fluorescence imaging image; e is the imaging image of the above fluorescent probe + ClO ⁻ bright field; f is the image after the above fluorescent probe ClO ⁻ bright field image and fluorescence image are superimposed.

Fig. 7 is the fluorescence imaging image of co-staining of pyrrole-merocyanine derivative fluorescent probe and commercial lysosome localization dye LysoTracker Red in HeLa cells; HeLa cells were co-stained with 1×10 ^-5 mol/L fluorescent probe and LysoTracker Red After 30 minutes of incubation, fluorescence imaging was performed using an Olympus FV500-IX70 laser confocal microscope.

Among them: a is the fluorescence image of the green channel; b is the fluorescence image of the red channel; c is the image after the green channel and the red channel are superimposed; d is the bright field image; e is the image after the green channel, the red channel and the bright field are superimposed ; f is an overlay of the intensity distributions of the green and red channels.

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments, but those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. The reagents and raw materials used in the embodiments of the present invention are obtained from conventional market purchases.

Example 1

The preparation method of the pyrrole-merocyanine derivative fluorescent probe in this embodiment is as follows:

Dissolve 2.79 g, N-morpholinoethyl-2,4-dimethyl-5-formylpyrrole-3-carboxamide (10 mmol) and 3.05 g N-ethylbenzothiazole iodonium salt (10 mmol) In 0.05L ethanol, 0.017 g piperidine (0.2 mmol) was added dropwise as a catalyst, refluxed and stirred at 80 °C for 3-4 h, cooled to room temperature, filtered under reduced pressure, the obtained solid was washed with ethanol, and then recrystallized with ethanol The pyrrole-merocyanine derivative fluorescent probe is obtained. The yield of the target product was 82%.

The obtained pyrrole-merocyanine derivatives were analyzed by nuclear magnetic resonance spectroscopy, and the results were as follows:

¹ H NMR (400 MHz, D ₂ O), δ (ppm): 7.85-7.87 (1H, d, Ar-H), 7.75-7.77 (1H, d, Ar-H), 7.60-7.63 (d, 1H , CH=), 7.57-7.61 (1H, t, Ar-H), 7.45-7.49 (1H, t, Ar-H), 6.92-6.96 (1H, d, CH=), 4.47-4.52 (2H, q , CH ₂ -CH ₃ ), 3.70-3.72(4H, t, 2CH ₂ ), 3.43-3.47 (2H, t, CH ₂ ), 2.62-2.64 (6H, m, 3CH ₂ ), 2.28 (3H, s, CH ₃ ), 2.17 (3H, s, CH ₃ ), 1.40-1.43 (3H, t, CH ₃ ). The specific H NMR spectrum is shown in Figure 1;

¹³ C NMR (400 MHz, DMSO- D ₆ ) Δ: 170.35, 164.33, 142.46, 141.13, 135.51,133.22, 129.44, 127.73, 126.71, 122.31, 115.84, 102.77,66.75, 57.69, 43.63, 43.63, 43.63, 43.63, 43. 36.26, 14.03, 13.59, 10.97. The specific carbon NMR spectrum is shown in Figure 2; the specific mass spectrum spectrum is shown in Figure 3.

Example 2

Determination of Optical Properties of Pyrrole-Merocyanine Derivatives for Hypochlorite

The pyrrole-merocyanine derivative prepared in the above Example 1 was used as a fluorescent probe to prepare a molar concentration of 1×10 ^-5 mol in a citric acid-sodium citrate buffer solution (0.02 mol/L, pH = 4). /L solution, respectively containing anions (AcO ⁻ , Br ⁻ , Cl ⁻ , ClO ⁻ , ClO ^{4 −} , CN − , F − , H 2 PO 4 − , ClO − , ClO ₄ − , CN ⁻ , F ⁻ , H ₂ PO ₄ ⁻ , An equal amount was added to the solution of HPO ₄ ⁻ , I ⁻ , PO ₄ ³⁻ , S ²⁻ and SO ₃ ²⁻ ) or reactive oxygen species (H ₂ O ₂ , ¹ O ₂ , OH, NO and O ₂ ⁻ ) The above fluorescent probe solution was analyzed by UV-Vis spectrophotometer or fluorescence spectrometer (excitation wavelength was 465 nm), and the obtained UV and fluorescence spectra were shown in Figure 4. It can be seen from Figure 4 that the pyrrole-merocyanine derivatives prepared by the present invention only have obvious responses to hypochlorite as a probe, and both ultraviolet and fluorescent signals can be used for rapid identification of hypochlorite, while other ions have no obvious response to hypochlorite. Variety.

The detection limit of ClO ⁻ is 1.65×10 ^-7 mol/L, and the linear detection ranges of UV and fluorescence spectra are 2.25×10 ^-5 -5.0×10 ^-5 mol/L and 2.25×10 -5 -5.0×10 -5 mol/L, respectively. 10.0×10 ^-5 -3.2×10 ^-5 mol/L, so the pyrrole-merocyanine derivatives prepared by the present invention can be used for the ultraviolet and fluorescence quantitative detection of hypochlorite.

Example 3

Fluorescent probes of pyrrole-merocyanine derivatives for the detection of hypochlorite in cells

HeLa cells were co-incubated with 1 × 10 ^-5 mol/L of the pyrrole-merocyanine derivative fluorescent probe prepared in Example 1 above and the commercial lysosome localization dye LysoTracker Red at 37°C for 30 minutes, and obtained in HeLa cells The fluorescence imaging image of the cell, as shown in Figure 6, in which: a is the fluorescence imaging image of the green channel; b is the fluorescence imaging image of the red channel; c is the superimposed image of the green channel and the red channel; d is the bright field image; e is the superimposed image of the green channel, the red channel and the bright field; f is the superimposed image of the intensity distribution of the green channel and the red channel. The fluorescence in the green channel of the probe and the fluorescence in the red channel of LysoTrackerRed in HeLa cells were basically consistent, and the overlap coefficient was 0.89. Therefore, the pyrrole-merocyanine derivative fluorescent probe prepared in Example 1 of the present invention can target cell lysosomes.

HeLa cells were incubated with 1×10 ^-5 mol/L of the pyrrole-merocyanine derivative fluorescent probe prepared in Example 1 above at 37°C for 30 minutes, and ClO ⁻ (1×10 ^-4 mol/L) was added. Incubate for another 30 minutes to obtain a fluorescence image of the HeLa cells, as shown in Figure 7, wherein: a is the green channel fluorescence image of the fluorescent probe; b is the bright field image of the fluorescent probe; c is the above-mentioned fluorescent probe. The superimposed image of the fluorescent probe brightfield image and the fluorescent image; d is the fluorescent imaging image of the above fluorescent probe + ClO ⁻ green channel; e is the imaging image of the above fluorescent probe + ClO ⁻ bright field; f is the above fluorescent probe The superimposed image of the needle ClO ⁻ brightfield image and the fluorescence image. The fluorescent probe of pyrrole-merocyanine derivatives showed strong fluorescence in HeLa cells, but the fluorescence decreased significantly after the addition of ClO ⁻ . Therefore, the pyrrole-merocyanine derivatives prepared in Example 1 of the present invention can be used for the qualitative detection of ClO ⁻ in cell lysosomes.

The above-mentioned embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope thereof is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention, and the protection scope of the present invention is subject to the claims.

Claims (8)

1. A pyrrole-merocyanine derivative fluorescent probe, wherein the pyrrole-merocyanine derivative fluorescent probe has the following structural formula:

. 2. the preparation method of pyrrole-merocyanine derivative fluorescent probe according to claim 1, is characterized in that, comprises the steps: S1: dissolve N-morpholinoethyl-2,4-dimethyl-5-formylpyrrole-3-carboxamide and N-ethylbenzothiazole iodonium salt in an organic solvent; S2: add piperidine dropwise to the solution obtained from S1 as a catalyst, and then stir the reaction at 80°C under reflux for 3-4h; S3: the solution obtained in S2 is cooled to room temperature, filtered under reduced pressure, the obtained solid residue is washed with ethanol, and then recrystallized with ethanol to obtain the pyrrole-merocyanine derivative fluorescent probe. 3 . The method for preparing a pyrrole-merocyanine derivative fluorescent probe according to claim 2 , wherein the organic solvent in the step S1 is anhydrous ethanol. 4 . 4. The method for preparing a pyrrole-merocyanine derivative fluorescent probe according to claim 2, wherein the N-morpholinoethyl-2,4-dimethyl-5 added in the step S1 -The molar ratio of formylpyrrole-3-carboxamide and N-ethylbenzothiazole iodonium salt is 1:1. 5 . The method for preparing a pyrrole-merocyanine derivative fluorescent probe according to claim 2 , wherein the time for the reflux stirring reaction in the step S2 is 3h. 6 . 6. The method for preparing a pyrrole-merocyanine derivative fluorescent probe according to claim 2, wherein in the step S2, N-morpholinoethyl-2,4-dimethyl-5- The molar ratio of formylpyrrole-3-carboxamide and piperidine was 1:0.02. 7. The preparation method of pyrrole-merocyanine derivative fluorescent probe according to claim 2, characterized in that the steps are as follows: 2.79 g of N-morpholinoethyl-2,4-dimethyl-5-methyl Acylpyrrole-3-carboxamide and 3.05 g of N-ethylbenzothiazole iodonium salt were dissolved in 0.05 L of ethanol, 0.017 g of piperidine was added dropwise as a catalyst, refluxed and stirred at 80 °C for 3-4 h, cooled and allowed to stand to room temperature, reduced The obtained solid is washed with ethanol and recrystallized with ethanol to obtain the pyrrole-merocyanine derivative fluorescent probe. 8. The application of the pyrrole-merocyanine derivative fluorescent probe according to claim 1 as a hypochlorite fluorescent probe in fluorescence imaging of cell lysosomes.

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