CN113149952B - A kind of chromene sulfonylhydrazone derivative fluorescent probe and its preparation method and application - Google Patents

Abstract

The present invention provides a chromene sulfonylhydrazone derivative fluorescent probe and its preparation method and application, wherein the chemical structural formula of the chromene sulfonylhydrazone derivative is as follows:

The preparation method is: dissolve 2H-benzene[h]chromene-3-formaldehyde and p-toluenesulfonyl hydrazide in ethanol; reflux and stir for 12 hours; after cooling to room temperature, filter, wash with ethanol, and dry to obtain yellow flaky crystals, It is the chromene sulfonylhydrazone derivative fluorescent probe; the chromene sulfonylhydrazone derivative fluorescent probe of the present invention can selectively interact with hypochlorite under physiological conditions, and the fluorescent color of the solution changes from blue to Turns green, showing a typical ratiometric fluorescence signal. Especially as a fluorescent probe for hypochlorite in fluorescent imaging of endoplasmic reticulum.

Description

A kind of chromene sulfonylhydrazone derivative fluorescent probe and its preparation method and application

technical field

The invention belongs to the field of organic synthesis, and specifically relates to a chromene sulfonylhydrazone derivative fluorescent probe and a preparation method and application thereof.

Background technique

Hypochlorous acid (HClO)/hypochlorite (ClO ⁻ ) is widely used as a disinfectant in daily life. At the same time, ClO ^- is also an important biological reactive oxygen species (ROS), involved in many physiological processes. However, its concentration exceeds the normal range and can cause various diseases including cancer. The endoplasmic reticulum is an important organelle, and its normal physiological function will be damaged when the cells are stimulated by chemical toxic substances, which is the so-called endoplasmic reticulum stress. Excessive or prolonged endoplasmic reticulum stress can induce apoptosis. The endoplasmic reticulum is one of the main sites for generating ROS in cells. Therefore, the detection and analysis of ClO ^- in the endoplasmic reticulum has very important research value, which will help to understand the apoptosis process induced by the endoplasmic reticulum.

Fluorescent probe technology has many advantages such as high sensitivity, simple operation, and real-time online detection, and is considered to be an ideal method for detecting ClO ^- . In recent years, some progress has been made in fluorescent probes for ^ClO- , and the design strategies usually use methoxyphenol, chalcogenides, hydrazides, hydroxylamine, or C=C as recognition groups, and utilize the strong oxidation of ^ClO- The specific oxidation of the recognition group will cause the fluorescence change of the probe to achieve the purpose of detection. However, most probes rely solely on the change of fluorescence intensity at a single emission wavelength to achieve detection, which is easily affected by environmental factors such as instrument efficiency and probe concentration. The ratiometric fluorescent probe uses the ratio of the intensity of two different emission peaks as the signal reference, which can effectively eliminate the above interference. Moreover, there are not many reports on ratiometric ClO ^- fluorescent probes targeting the endoplasmic reticulum.

In view of this, the present invention is proposed.

Contents of the invention

Aiming at the problems existing in the prior art, considering that 2H -benzo[ h ]chromene-3-carbaldehyde has excellent optical properties, and sulfonyl hydrazone can be used as the recognition unit of ^ClO- , the present invention uses chromenesulfonyl A highly sensitive and highly selective ratiometric ClO ^- fluorescent probe was synthesized based on hydrazone derivatives. The probe has endoplasmic reticulum targeting function and can be used to detect the ClO ^- concentration in the cell endoplasmic reticulum.

The main purpose of the present invention is to provide a kind of chromene sulfonylhydrazone derivative fluorescent probe that can be used in the cell endoplasmic reticulum for ClO ^- high sensitivity and good selectivity; Another purpose is to provide the fluorescent probe of this fluorescent probe Preparation methods and applications.

In order to achieve the above object, the present invention adopts the following technical scheme: a fluorescent probe of chromene sulfonylhydrazone derivatives, the chromene sulfonylhydrazone derivatives have the following structural formula:

.

The present invention also provides a preparation method of a chromene sulfonylhydrazone derivative fluorescent probe, the specific preparation method is as follows:

S1: Dissolving 2H-benzene[h]chromene-3-carbaldehyde and p-toluenesulfonyl hydrazide in ethanol;

S2: Reflux and stir for 12h;

S3: After cooling to room temperature, filter, wash with ethanol, and dry to obtain yellow flaky crystals, which are the fluorescent probes of chromenesulfonylhydrazone derivatives.

Furthermore, the ethanol in steps S1 and S3 is 95% ethanol or absolute ethanol.

Furthermore, the molar ratio of 2H-benzo[h]chromene-3-carbaldehyde to p-toluenesulfonylhydrazide added in step S1 is 1:1.

Furthermore, the specific preparation method is that a mixture of 2H-benzene[h]chromene-3-carbaldehyde (210 mg, 1 mmol) and p-toluenesulfonyl hydrazide (190 mg, 1 mmol) was added to 10 mL of ethanol In, reflux and stir for 12h. After cooling to room temperature, filter, wash with ethanol, and dry to obtain yellow flaky crystals, which are the fluorescent probes of chromenesulfonylhydrazone derivatives.

The present invention also provides the use of the fluorescent probe of the chromene sulfonylhydrazone derivative, that is, the application as a ^ClO- fluorescent probe, especially the application as a fluorescent probe for detecting ^ClO- in the cell endoplasmic reticulum.

Compared with prior art, advantage and positive effect of the present invention are:

The invention prepares the chromene sulfonylhydrazone derivative fluorescent probe through the condensation reaction, the raw materials are easy to obtain, and the synthesis and post-treatment methods are simple. Among a variety of common anions and reactive oxygen species, ClO ^- exhibits higher ratiometric fluorescence recognition performance. The probe can target the endoplasmic reticulum of cells and has a wide range of potential applications.

Description of drawings

Fig. 1 is the ¹ H NMR spectrogram of the chromene sulfonylhydrazone derivative fluorescent probe prepared in Example 1 of the present invention;

Fig. 2 is the ¹³ C NMR spectrogram of the chromene sulfonylhydrazone derivative fluorescent probe prepared in Example 1 of the present invention;

Fig. 3 is the mass spectrogram of the chromene sulfonylhydrazone derivative fluorescent probe prepared in Example 1 of the present invention;

Fig. 4 is the crystal structure diagram of the chromene sulfonylhydrazone derivative fluorescent probe prepared in Example 1 of the present invention;

Figure 5 shows the DMSO/PBS (2/8, v/v, ¹⁰ mM, pH=7.0 ) solution was added with 5×10 ^-4 mol/L anions (HSO ₃ ^- , SO ₄ ^2- , PPi, HSO ₄ ^- , PO ₄ ^3- , H ₂ PO ₄ ^- , CN ^- , ClO ₄ ^- , HPO ₄ ^{2 -} , I ^- , Br ^- , F ^- , S ^2- , Cl ^- , OAC ^- , SO ₃ ^2- ) and reactive oxygen species (ClO ^- , H ₂ O ₂ , ¹ O ₂ , ·OH, NO and O ₂ ⁻ ) fluorescence spectrum (excitation wavelength is 400 nm), the inset shows the color change of probe and probe + ClO ^- solution under 365 nm UV lamp;

Figure 6a is the DMSO/PBS (2/8, v/v, ¹⁰ mM, pH=7.0 ) solution titrated with different concentrations of ClO ⁻ fluorescence spectra (excitation wavelength is 400 nm). Figure 6b shows the linear change trend graph of the fluorescence intensity ratio at 525 nm and 475 nm with the concentration of ^ClO- .

Figure 7 is the co-staining fluorescence imaging image of chromene sulfonylhydrazone derivative fluorescent probe and commercial ^endoplasmic reticulum localization dye ERTracker Red in HeLa cells; After 30 min of Red co-incubation, fluorescence imaging was performed using an Olympus FV500-IX70 confocal laser microscope.

Among them: a is the fluorescence imaging image of the blue channel; b is the fluorescence imaging image of the red channel; c is the superimposed picture of the blue channel and the red channel; d is the bright field image; e is the superposition of the blue channel, the red channel and the bright field The picture after; f is the overlay of the intensity distribution of the blue channel and the red channel.

Figure 8 is the fluorescence imaging image of chromene sulfonylhydrazone 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 with 5×10 ^-4 mol/L ClO ⁻ , after further incubation for 30 minutes, fluorescence imaging was performed using an Olympus FV500-IX70 laser confocal microscope.

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

Detailed ways

The present invention will be further described in 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 examples of the present invention were purchased from conventional markets.

Example 1

The reaction scheme of the chromene sulfonylhydrazone derivative fluorescent probe in this embodiment is as follows:

.

The preparation method of the chromene sulfonylhydrazone derivative fluorescent probe of this embodiment is as follows:

Add a mixture of 2H-benzene[h]chromene-3-carbaldehyde (210 mg, 1 mmol) and p-toluenesulfonylhydrazide (190 mg, 1 mmol) into 10 mL of ethanol, and stir at reflux for 12 hours. After cooling to room temperature, filter, wash with ethanol, and dry to obtain yellow flaky crystals with a yield of 86%. Adopt nuclear magnetic resonance instrument to carry out nuclear magnetic resonance analysis to the chromene sulfonylhydrazone derivative that makes, the result is as follows:

¹ H NMR (400 MHz, d ₆ -DMSO) δ (ppm): 11.5 (1H, s, NH), 8.13-8.15 (1H, s, CH=N), 7.76-7.86 (5H, m) / 7.68 ( 1H, s) / 7.53-7.56 (1H, s) /7.39-7.45(3H, s) for Ar-H, 7.12-7.14 (1H, s, CH), 4.98 (2H, s, CH ₂ ), 2.39 ( 3H, s, CH ₃ ). The specific hydrogen NMR spectrum is shown in Figure 1;

¹³ C NMR (400 MHz, d ₆ -DMSO) δ (ppm): 153.09, 146.32, 144.00, 136.37, 131.32, 130.15, 129.50, 129.00, 127.69, 127.32, 125.43, 124.62, 122.16, 117.78, 115.14, 63.94, 21.50 . The specific carbon NMR spectrum is shown in Figure 2;

Mass Spectrum ESI-MS: m/z = 379.1079 for [M+H] ⁺ . See Figure 3 for the specific mass spectrogram.

Single crystal X-ray diffraction confirmed the crystal structure of the probe, see Figure 4.

Example 2

Determination of Optical Properties of Chromene Sulfonylhydrazone Derivatives to ClO ^-

The chromene sulfonylhydrazone derivative prepared in Example 1 above was used as a fluorescent probe in DMSO/PBS (2/8, v/v, 10 mM, pH=7.0) to a molar concentration of 1×10 ^-5 mol/L solution, respectively in the anions (HSO ^{3 -} , SO ₄ ^2- _{, PPi, HSO 4 - , PO 4 3-} ^, _H ² _{PO 4} ^- , CN ^- , ClO ₄ ^- , HPO ₄ ^2- , I ^- , Br ^- , F ^- , S ^2- , Cl ^- , OAC ^- , SO ₃ ^2- ) and reactive oxygen species (ClO ^- , H ₂ O ₂ , ¹ O ₂ , ·OH, NO and O ₂ ⁻ ) solutions were added with the same amount of the above-mentioned fluorescent probe solution, and the fluorescence spectrometer was used for analysis (excitation wavelength was 400 nm). The obtained fluorescence spectrum is shown in Figure 5. As can be seen from Figure 5, only ^ClO- can cause the emission peak of the probe to be enhanced and red-shifted, while other ions do not change, indicating that the chromene sulfonylhydrazone derivatives prepared by the present invention only have a significant response to ^ClO- as probes . The inset shows the color change of the probe and probe+ClO ^- solution under 365 nm ultraviolet light. It can be seen that the probe solution changes from blue to green after adding ClO ^- , indicating that the probe can be used for rapid identification of ClO ^- .

According to the titration spectrum calculation in Figure 6, it can be obtained that the detection limit of ClO ⁻ is 1.01×10 ^-8 mol/L, and the linear detection range of the fluorescence intensity ratio F ₅₂₅ /F ₄₇₅ is 3.0×10 ^-6 -8.0×10 ^-6 mol/L L. Therefore, the chromene sulfonylhydrazone derivatives prepared in the present invention can be used for the fluorescence quantitative detection of ^ClO- .

Example 3

Chromene Sulfonylhydrazone Derivative Fluorescent Probes for Detection of ClO ^- in Cells

HeLa cells were co-incubated with 1×10 ^-5 mol/L of the chromene sulfonylhydrazone derivative fluorescent probe prepared in Example 1 above and the commercial lysosome localization dye ER Tracker Red at 37°C for 30 minutes to obtain The fluorescence imaging image of HeLa cells is shown in Figure 7, in which: a is the fluorescence imaging image of the blue channel; b is the fluorescence imaging image of the red channel; c is the superimposed picture of the blue channel and the red channel; d is the bright field Figure; e is the superimposed image of the blue channel, red channel and bright field; f is the superimposed image of the intensity distribution of the blue channel and the red channel. The blue channel fluorescence of the probe in HeLa cells and the red channel fluorescence of ER Tracker Red are basically consistent, with an overlap coefficient of 0.88. Therefore, the chromene sulfonylhydrazone derivative fluorescent probe prepared in Example 1 of the present invention can target the endoplasmic reticulum of cells.

HeLa cells were incubated with 1×10 ^-5 mol/L fluorescent probe of chromene sulfonylhydrazone derivatives prepared in Example 1 above at 37°C for 30 minutes, and ClO ⁻ (5×10 ^-4 mol/L) was added After incubation for another 30 minutes, the fluorescence imaging diagram of the HeLa cells was obtained, as shown in Figure 8, wherein: a is the fluorescence imaging diagram of the blue channel of the above-mentioned fluorescent probe; b is the fluorescence imaging diagram of the green channel of the above-mentioned fluorescent probe; c is the bright-field image of the above-mentioned fluorescent probe; d is the superimposed picture of the bright-field image and the fluorescence image of the above-mentioned fluorescent probe; e is the fluorescence imaging image of the above-mentioned fluorescent probe + ClO ⁻ blue channel; f is the above-mentioned fluorescent probe + ClO ^- Fluorescence imaging image of the green channel; g is the imaging image under the above fluorescent probe + ClO ⁻ bright field; h is the superimposed image of the above fluorescent probe + ClO ⁻ bright field image and fluorescence image. Adding chromene sulfonylhydrazone derivatives to HeLa cells showed strong fluorescence in the blue channel and weak fluorescence in the green channel; however, after adding ClO ⁻ the fluorescence in the blue channel was significantly weakened and the fluorescence in the green channel was significantly enhanced. Therefore, the chromene sulfonylhydrazone derivatives prepared in Example 1 of the present invention can be used for ratiometric fluorescence detection of ClO ⁻ in the endoplasmic reticulum of cells.

The above-mentioned embodiments are only preferred embodiments for fully illustrating the present invention, and the scope of protection thereof is not limited thereto. The 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 shall be determined by the claims.

Claims (6)

1. a chromene sulfonylhydrazone derivative fluorescent probe, characterized in that, the chromene sulfonylhydrazone derivative fluorescent probe has the following structural formula: . 2. the preparation method of chromene sulfonylhydrazone derivative fluorescent probe according to claim 1, is characterized in that, comprises the steps: S1: dissolving compound 1 and p-toluenesulfonyl hydrazide in ethanol; S2: Reflux and stir for 12h; S3: After cooling to room temperature, filter, wash with ethanol, and dry to obtain yellow flaky crystals, which are the fluorescent probes of chromenesulfonylhydrazone derivatives; The structural formula of the compound 1 is as follows: . 3. The preparation method of the chromene sulfonylhydrazone derivative fluorescent probe according to claim 2, characterized in that: the ethanol in the steps S1 and S3 is 95% ethanol or absolute ethanol. 4. The preparation method of the chromenesulfonylhydrazone derivative fluorescent probe according to claim 2, characterized in that: the molar ratio of the compound 1 added in the step S1 to p-toluenesulfonylhydrazide is 1:1. 5. the preparation method of chromene sulfonylhydrazone derivative fluorescent probe according to claim 2 is characterized in that concrete steps are as follows: 1 mmol compound 1 and 1 mmol p-toluenesulfonyl hydrazide are added in the ethanol of 10 mL , stirred at reflux for 12 h, cooled to room temperature, filtered, washed with ethanol, and dried to obtain yellow flaky crystals, which are the fluorescent probes of chromenesulfonylhydrazone derivatives. 6. The application of the chromene sulfonylhydrazone derivative fluorescent probe in the preparation of hypochlorite fluorescent probe according to claim 1, characterized in that: the fluorescent probe is used for fluorescence imaging of endoplasmic reticulum.

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