CN110878049A - Preparation and application of fluorescent probe for specifically analyzing hydrogen sulfide in Golgi apparatus - Google Patents

Abstract

The invention relates to preparation and application of a fluorescent probe for specifically analyzing hydrogen sulfide in a Golgi apparatus, in particular to a fluorescent probe for naphthalimide compounds, which can be used as a hydrogen sulfide fluorescent probe, especially can be used for targeting positioning of the Golgi apparatus and is used for measuring, detecting or screening the hydrogen sulfide in the Golgi apparatus. Such probes can achieve at least one of the following technical effects: the selectivity is high, and hydrogen sulfide in a Golgi apparatus can be measured, detected or screened; the anti-interference capability is strong, and the interference of other substances in the life body corresponding to the detection of the probe can be prevented; the sensitivity is high, and the method is suitable for detecting trace hydrogen sulfide in a living body; simple synthesis and stable property, and is suitable for commercial popularization and use.

Description

Preparation and application of a fluorescent probe for the specific analysis of hydrogen sulfide in the Golgi apparatus

technical field

The invention belongs to the field of fluorescent probes, and in particular relates to a fluorescent probe of a naphthalimide compound and its application in measuring, detecting or screening hydrogen sulfide in the Golgi apparatus and a live cell fluorescent imaging method; the invention also provides A method for preparing the fluorescent probe is presented.

Background technique

Hydrogen sulfide is a colorless gas with irritating and suffocating properties. Low concentration exposure only has local irritating effects on the respiratory tract and eyes. At high concentrations, the systemic effects are more obvious, manifesting as symptoms of the central nervous system and suffocation. Hydrogen sulfide has "odorous" "Egg" smell, but very high concentration of hydrogen sulfide will quickly cause olfactory fatigue without feeling its taste. Hydrogen sulfide will have a strong stimulating effect on the eyes and respiratory mucosa. After absorption of hydrogen sulfide, it mainly affects the process of cell oxidation and causes tissue deficiency. Oxygen. Mild cases are mainly irritation symptoms, manifested as tearing, eye irritation, runny nose, burning sensation in the throat, or accompanied by symptoms such as headache, dizziness, fatigue, nausea, etc. In addition, endogenous hydrogen sulfide has been proved to be a It also provides cytoprotective agents, neuroprotective agents, anti-inflammatory agents, antioxidants, apoptotic agents, etc.

In view of this, it is extremely important and meaningful to develop analytical methods that can effectively detect fluctuations in H2S content, especially in specific organelles. The reported analytical methods for detecting hydrogen sulfide include mercury quantification, detection tube method, and methylene blue colorimetry. Among these numerous detection methods, fluorescent probes have become the focus of researchers due to their unique advantages. However, the currently reported fluorescent probes still have some problems, including insufficient selectivity, insufficient response speed, complex synthesis, and inability to detect and analyze hydrogen sulfide in specific organelles. Due to other components in the body such as alanine (Ala), phenylalanine (Phe), methionine (Met), glycine (Gly), glutamic acid (Glu), arginine (Arg), lysine ( Lys), tryptophan (Trp), sulfate (SO ₄ ^2- ), hydrogen sulfite (HSO ₃ ^- ), chloride (Cl ^- ), carbonate (CO ₃ ^2- ), bicarbonate (HCO ₃ ^- ), nitrate (NO ₃ ^- ), sulfite (SO ₃ ^2- ), hydrogen peroxide (H ₂ O ₂ ), hypochlorite (ClO ^- ), etc., which may interfere with the detection of hydrogen sulfide Therefore, the rapid development, high selectivity, high sensitivity, simple synthesis, and the ability to measure, detect or screen hydrogen sulfide fluorescent probes in the Golgi apparatus has become an urgent problem for those skilled in the art.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to provide a kind of fluorescent probes for highly selective analysis of hydrogen sulfide in the Golgi apparatus, as well as their preparation methods and uses, which have the advantages of simple synthesis, good selectivity, high sensitivity, and capable of measuring and detecting Or screen for the characteristics of hydrogen sulfide in the Golgi apparatus.

Specifically, the present invention provides a compound having the structure shown in formula (I):

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ and R ₉ are hydrogen atoms, straight-chain or branched-chain alkyl groups, straight-chain or branched-chain alkoxy groups, sulfonic acid groups , ester group, carboxyl group; R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ and R ₉ may be the same or different.

In some specific embodiments of the present invention, the compound of the present invention is of formula (I) wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ and R ₉ are all hydrogen atoms compound, its structural formula is as follows:

The present invention also provides a method for preparing the compound of formula (I) or formula (II), comprising the following steps: dissolving the compound of formula (III) and sodium azide in dimethyl sulfoxide (DMSO), then heating and stirring until The reaction finishes, and the reaction solution is subjected to extraction and suction filtration to obtain a crude product, which is separated and purified to obtain a pure compound of formula (I), and its reaction formula is as follows:

Wherein: R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ and R ₉ are hydrogen atoms, linear or branched alkyl, linear or branched alkoxy, sulfonic Acid group, ester group, carboxyl group; R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ and R ₉ may be the same or different.

In some specific embodiments of the present invention, the reaction time is 6-48 hours.

In some specific embodiments of the present invention, the molar ratio of the compound of formula (III) to sodium azide is 1:1-10:1.

In some specific embodiments of the present invention, the heating reaction temperature is 80-100°C.

In some specific embodiments of the present invention, the separation and purification method is chromatographic column separation.

In some specific embodiments of the present invention, the eluent used for the chromatographic column separation is a mixed solvent of dichloromethane and petroleum ether.

In some specific embodiments of the present invention, compounds of formula (III) wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ and R ₉ are hydrogen atoms are combined with azide Sodium is dissolved in dimethyl sulfoxide (DMSO), the molar ratio of the two is 1:1-1:5, and then heated and stirred for 10-16 hours. The reaction solution is extracted and filtered to obtain a crude product, and the crude product is used as an eluent with a mixed solvent (volume ratio of 1:1-1:3) of dichloromethane and petroleum ether, and purified and separated by a chromatographic column to obtain a pure formula ( I) Compounds.

The present invention also provides a fluorescent probe composition for measuring, detecting or screening hydrogen sulfide, comprising the compound of formula (I) of the present invention.

In some specific embodiments of the present invention, the fluorescent probe composition has the following structure:

In some specific embodiments of the present invention, the fluorescent probe composition further comprises a solvent, an acid, a base, a buffer solution, or a combination thereof.

The present invention also provides a method for detecting the presence of hydrogen sulfide in the sample or determining the hydrogen sulfide content in the sample, comprising:

a) contacting the compound of formula (I) or formula (II) with a sample to form a fluorescent compound;

b) Determining the fluorescent properties of the fluorescent compound.

In some embodiments of the invention, the sample is a chemical sample or a biological sample.

In some embodiments of the invention, the sample is a biological sample including water, blood, microorganisms, or animal cells or tissues.

The present invention also provides a kit for detecting the presence of hydrogen sulfide in a sample or determining the hydrogen sulfide content in a sample, comprising the compound of formula (I) or formula (II).

The present invention also provides the application of the compound of formula (I) or formula (II) in cell fluorescence imaging.

The present invention also provides the application of the compound of formula (I) or formula (II) in measuring, detecting or screening hydrogen sulfide by targeting the Golgi apparatus.

Compared with the prior art, the present invention has the following significant advantages and effects: high selectivity, capable of specifically identifying hydrogen sulfide in the Golgi apparatus; strong anti-interference ability, capable of preventing other substances in the living body from interfering with the detection of the probe; High sensitivity, suitable for the detection of trace hydrogen sulfide in living body; simple synthesis, stable properties, suitable for commercial promotion.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained according to these drawings without creative efforts.

Figure 1(a) Fluorescence spectra of the probe (5 μM) before and after adding hydrogen sulfide (0-100 μM);

Figure 1(b) The working curve of the probe (5μM) for quantitative analysis of different concentrations of hydrogen sulfide (0-30μM);

Fig. 2 The effect of common substances in the human body on the fluorescence intensity of the probe (5 μM). The histograms represent the fluorescence intensity values of probes at 550 nm in the presence of different analytes;

Figure 3. Cells were incubated with probes and different commercial organelle dyes, and images were acquired by confocal microscopy.

Detailed ways

The technical solutions in the embodiments of the present invention will be described clearly and completely below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention and should not be used to limit the present invention. protected range. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

Example 1: Synthesis of compound of formula (II)

The synthetic design route is as follows:

Embodiment 1: Dissolve 386 mg (1 mmol) of the compound of formula (IV) and 65 mg (1 mmol) of sodium azide in DMSO, heat and stir at 90° C. for 10 h, extract and suction the reaction solution to obtain a crude product, and use the two A mixed solvent of methyl chloride and petroleum ether (volume ratio of 1:1) is used as an eluent, and purified and separated by a chromatographic column to obtain a pure compound of formula (II). A dark yellow color was obtained to obtain 235 mg of a dark yellow compound of formula (II) with a yield of 60%.

Embodiment 2: Dissolve 386 mg (1 mmol) of the compound of formula (IV) and 130 mg (2 mmol) of sodium azide in DMSO, heat and stir at 90° C. for 11 h, extract and filter the reaction solution to obtain a crude product. A mixed solvent of methyl chloride and petroleum ether (volume ratio of 1:1) is used as an eluent, and purified and separated by a chromatographic column to obtain a pure compound of formula (II). 247 mg of the compound of formula (II) in dark yellow was obtained in a yield of 63%.

Embodiment 3: Dissolve 386 mg (1 mmol) of the compound of formula (IV) and 195 mg (3 mmol) of sodium azide in DMSO, heat and stir at 90° C. for 12 h, extract and suction the reaction solution to obtain a crude product, and use the two A mixed solvent of methyl chloride and petroleum ether (volume ratio of 1:1) is used as an eluent, and purified and separated by a chromatographic column to obtain a pure compound of formula (II). 255 mg of the compound of formula (II) in dark yellow was obtained in a yield of 65%.

Embodiment 4: Dissolve 386 mg (1 mmol) of the compound of formula (IV) and 260 mg (4 mmol) of sodium azide in DMSO, heat and stir at 90° C. for 13 h, extract and suction the reaction solution to obtain a crude product, and use the two A mixed solvent of methyl chloride and petroleum ether (volume ratio of 1:1) is used as an eluent, and purified and separated by a chromatographic column to obtain a pure compound of formula (II). 267 mg of the dark yellow compound of formula (II) was obtained with a yield of 68%.

Embodiment 5: Dissolve 386 mg (1 mmol) of the compound of formula (IV) and 325 mg (5 mmol) of sodium azide in DMSO, heat and stir at 90° C. for 14 h, extract and suction the reaction solution to obtain a crude product. A mixed solvent of methyl chloride and petroleum ether (volume ratio of 1:1) is used as an eluent, and purified and separated by a chromatographic column to obtain a pure compound of formula (II). 290 mg of the compound of formula (II) in dark yellow was obtained in a yield of 74%.

Example 2: Testing the concentration gradient of fluorescent probes for hydrogen sulfide

Arrange multiple parallel samples with a probe concentration of 5 μM in a 10 mL colorimetric tube, then add different concentrations of hydrogen sulfide (0-100 μM) into the test system, shake evenly, and let stand for 30 min. The above assays were performed in a pure water system (0.5 mM PBS, pH 7.4), the probes used were those prepared in Example 1, and all spectroscopic measurements were performed at 25°C.

The fluorescence intensity changes were measured with a fluorescence spectrometer. It can be clearly seen from Figure 1a that with the increase of hydrogen sulfide concentration, the fluorescence intensity at 550 nm gradually increased. Moreover, it can be seen from Figure 1b that the fluorescence intensity shows a good linear relationship after the addition of hydrogen sulfide (0-30 μM) to the fluorescent probe (5 μM), which proves that the fluorescent probe can be used for quantitative analysis of hydrogen sulfide.

Example 3: Testing the selectivity of fluorescent probes for hydrogen sulfide

Analytes include: 1 blank probe (5μM), 2 potassium ions (K ⁺ ), 3 calcium ions (Ca ²⁺ ), 4 sodium ions (Na ⁺ ), 5 fluoride ions (F ^- ), 6 chloride ions (Cl ) ^- ), 7 bromide (Br ^-) , 8 phenylalanine (Phe), 9 methionine (Met), 10 glycine (Gly), 11 glutamic acid (Glu), 12 arginine (Arg), 13 lysine Amino acid (Lys), 14 Tryptophan (Trp), 15 Serine (Ser), 16 Threonine (Thr), 17 Aspartic acid (Asp), 18 Proline (Pro), 19 Leucine ( Leu), 20 phosphate (PO ₄ ^3- ), 21 sulfate (SO ₄ ^2- ), 22 cysteine (Cys), 23 glutathione (GSH), 24 hydrogen sulfide (100μM), unless otherwise specified The concentrations of exoanalytes were all 1 mM. All test conditions were done in pure water system (0.5mM PBS, pH 7.4), the probe used was the probe prepared in Example 1, and all spectra were measured at 25°C after analyte addition for 30 minutes Got it. Specifically, a part of water was added first, then 0.5 mL of PBS7.4 (10 mM) buffer solution was added, 100 μL of the above-mentioned analyte (2-23) stock solution (100 mM) was then added to the tube, and finally the volume was adjusted to 10 mL with water. The results are shown in Figure 2, which collects the fluorescence intensity at the emission wavelength of 550 nm. It can be seen from Figure 2 that the probe has good selectivity and can specifically recognize hydrogen sulfide.

Example 4: Labeling of Different Cells with Probes and Different Commercial Organelle Dyes

HeLa cells were first incubated with hydrogen sulfide (100 μM) for 30 minutes, then probes (10 μM) and commercial organelle dyes (mitochondria, endoplasmic reticulum, lysosomes, Golgi) were added for 30 minutes of incubation followed by phosphate buffered saline. Washed 3 times to reduce background fluorescence and imaged with a confocal microscope. Green channel excitation wavelength 488nm, collection wavelength 510-590nm; red channel: mitochondria excitation wavelength 578nm, collection wavelength 590-640nm; endoplasmic reticulum excitation wavelength 594nm, collection wavelength 600nm -670nm; lysosome excitation wavelength 559nm, collection wavelength 585-620nm; Golgi emission wavelength 633nm, collection wavelength 634-740nm. As can be seen from Figure 3, the probe has strong tissue penetration and can detect hydrogen sulfide in cells. By comparing with dyes such as Golgi, the superimposed field and the red-green fluorescence overlap coefficient map (A4-D4) It can be seen that the probe has the best overlap effect with the Golgi apparatus, and the overlap coefficients are mitochondria (0.26), endoplasmic reticulum (0.18), lysosome (0.33), and Golgi apparatus (0.94), showing that the probe is an excellent target. the ability to locate the Golgi apparatus.

Although the present invention has been described with the above embodiments, it should be understood that, without departing from the spirit of the present invention, the present invention can be further modified and changed, and these modifications and changes all fall within the protection scope of the present invention .

Claims (10)

1. A compound having the structure:

wherein: r₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈And R₉Is hydrogenAtom, straight chain or branched chain alkyl, straight chain or branched chain alkoxy, sulfonic group, ester group and carboxyl; r₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈And R₉May be the same or different.

2. A compound according to claim 1, characterized in that R₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈And R₉Are all hydrogen atoms.

3. A process for the preparation of a compound according to claim 1 or 2, comprising the steps of: dissolving a compound shown in a formula (III) and sodium azide in dimethyl sulfoxide (DMSO), heating and stirring until the reaction is finished, extracting and filtering reaction liquid to obtain a crude product, and separating and purifying to obtain a pure compound shown in a formula (I), wherein the reaction formula is as follows:

wherein: r₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈And R₉Is hydrogen atom, straight chain or branched chain alkyl, straight chain or branched chain alkoxy, sulfonic group, ester group, carboxyl; r₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈And R₉May be the same or different.

4. The method of claim 3, comprising the steps of:

r is to be₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈And R₉Dissolving a compound of formula (III) and sodium azide, both hydrogen atoms, in dimethyl sulfoxide (DMSO) in a molar ratio of 1:1 to 1:5, and addingStirring under heat for 6-48 hours. And (3) extracting and filtering the reaction liquid to obtain a crude product, and purifying and separating the crude product by using a mixed solvent of dichloromethane and petroleum ether as an eluent through a chromatographic column to obtain the pure compound shown in the formula (I).

5. A fluorescent probe composition for measuring, detecting or screening hydrogen sulfide, comprising the compound of any one of claims 1-2.

6. The fluorescent probe composition of claim 5, wherein the compound is:

7. the fluorescent probe composition of claim 5 or 6, wherein the fluorescent probe composition further comprises a solvent, an acid, a base, a buffer solution, or a combination thereof.

8. A method for detecting the presence of or determining the amount of hydrogen sulfide in a sample, comprising:

a) contacting a compound of any one of claims 1-2 with a sample to form a fluorescent compound;

b) determining the fluorescent properties of the fluorescent compound.

9. Use of a compound according to any one of claims 1-2 in fluorescence imaging of cells.

10. Use of a compound according to any one of claims 1-2 for the targeted localization of golgi to measure, detect or screen for hydrogen sulfide.

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