A fluorescent probe for detecting H2S gas signal molecules and its preparation method and application

Abstract

The present invention discloses a method for detecting H 2 S gas signal molecule fluorescent probe and its preparation method and application. The fluorescent probe has the following structural expression. The preparation method of the fluorescent probe comprises the following steps: 1) dissolving 4-bromo-1,8-naphthalene dicarboxylic anhydride and 4-(2-aminoethyl)-thiomorpholine in solvent A, stirring and reacting under reflux conditions to obtain an intermediate product; 2) mixing the intermediate product and sodium azide in solvent B, stirring and reacting under reflux conditions to generate the target product fluorescent probe. The preparation method of the fluorescent probe is simple, and it reacts with H at body temperature. 2 S responds quickly, has strong fluorescence, and has high detection sensitivity. #imgabs0#

Description

Technical Field

[0001] The invention relates to a fluorescent probe, and in particular to a fluorescent probe for detecting H2S gas signal molecules and a preparation method and application thereof. Background Art

[0002] Hydrogen sulfide (H2S) is an important endogenous gaseous signaling molecule that plays an important role in regulating organelle function and stress. It is the third most important gasotransmitter regulating the cardiovascular, nervous, immune, endocrine and gastrointestinal systems. Endogenous H2S levels are thought to be associated with certain diseases, such as Alzheimer's disease, Down syndrome, diabetes and cirrhosis. H2S inhibitors and H2S donors in animal disease models have shown potential for therapeutic utilization of H2S. Therefore, it will be very important to visualize the distribution and concentration of H2S in biological systems to help elucidate the biological role of H2S.

[0003] Fluorescence imaging in living cells is an imaging technology for studying various biochemical activities in organisms. By attaching subcellular organelle specific groups, fluorescent probes can detect target analytes and reveal various biochemical activities in specific areas of cells. It has many advantages such as high sensitivity, real-time imaging and high spatiotemporal resolution, and therefore has great application potential.

[0004] So far, researchers have reported a variety of fluorescent probes for efficient hydrogen sulfide detection. Commonly used fluorescent dyes include rhodamine, coumarin series, fluorescein, naphthalimide and boron dipyrrolidine. 1,8-Naphthalimide is a typical D-π-A fluorescent dye with the advantages of stable optical properties, high fluorescence quantum yield, strong fluorescence, and long excitation and emission wavelengths. It is widely used in the development of hydrogen sulfide fluorescent probes. For example:

[0005] Patent CN113024463A discloses the preparation and application of a 1,8-naphthalimide-based hydrogen sulfide fluorescent molecular probe. Using 4-bromo-1,8-naphthalene anhydride and 4-aminophenol as raw materials, a 1,8-naphthalene imide-based hydrogen sulfide fluorescent molecular probe was prepared through a four-step reaction. Patent CN114874142A discloses a method for preparing a fluorescent probe based on 1,8-naphthalene imide derivatives through a three-step reaction using 4-bromo-1,8-naphthalene anhydride and dimethyldiamine as raw materials. The recognition of hydrogen sulfide can achieve the effect of naked eye recognition. Patent CN104610959B discloses a fluorescent probe for detecting hydrogen sulfide from naphthalene imide and p-azidobenzyl alcohol to obtain a "ratiometer-type" fluorescence response, which greatly improves the sensitivity of detection.

[0006] However, not all fluorescent probes have the characteristics of sensitive H2S detection, high fluorescence intensity and good biocompatibility at in vivo temperature. Therefore, it is still of great research significance to develop fluorescent probes of hydrogen sulfide gas signal molecules with novel structures and simple preparation processes to make them more suitable for detecting endogenous H2S gas signal molecules in living cells. Summary of the invention

[0007] In order to solve the above technical problems, the present invention first proposes a fluorescent probe for detecting H2S gas signal molecules. The fluorescent probe responds quickly to H2S at body temperature, exhibits strong fluorescence, and has high detection sensitivity.

[0008] Based on the second aspect of the present invention, a method for preparing a fluorescent probe for detecting H2S gas signal molecules is also proposed. The present invention prepares a fluorescent probe based on 1,8-naphthalimide derivatives through only two steps, which has the advantages of simple process and low cost.

[0009] Based on the third aspect of the present invention, an application of a fluorescent probe for detecting H2S gas signal molecules in living cell fluorescence imaging is also proposed, especially as a targeted fluorescent probe for endogenous H2S gas signal molecules.

[0010] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0011] The present invention first provides a fluorescent probe for detecting H2S gas signal molecules, wherein the fluorescent probe has the following structural expression:

[0012]

[0013] The present invention designs the molecular structure of the fluorescent probe, uses a naphthalimide derivative as a fluorescent group, an azide group as a recognition group, and 4-(2-aminoethyl)-thiomorpholine as a targeting group, so as to achieve high-sensitivity recognition of H2S gas signal molecules, a low detection limit, and surprisingly, the fluorescence enhancement and sensitive detection characteristics can still be maintained at the body temperature. In addition, 4-(2-aminoethyl)-thiomorpholine is non-toxic and harmless, has good biocompatibility, and can ensure that the fluorescent probe synthesized with it as a raw material has low cytotoxicity.

[0014] The present invention also provides a method for preparing a fluorescent probe for detecting H2S gas signal molecules, comprising the following steps:

[0015]

[0016] 1) dissolving 4-bromo-1,8-naphthalene dicarboxylic anhydride and 4-(2-aminoethyl)-thiomorpholine in solvent A, stirring and reacting under reflux conditions to obtain an intermediate product;

[0017] 2) The intermediate product and sodium azide are mixed in solvent B, and reacted by stirring under reflux conditions to generate the target product fluorescent probe.

[0018] As a preferred embodiment of the preparation method of the present invention, in step 1), the molar ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride to 4-(2-aminoethyl)-thiomorpholine is 1:(1-2.5), for example, 1:1, 1:1.25, 1:1.5, 1:1.8, 1:2, 1:2.3, etc.

[0019] As a preferred embodiment of the preparation method of the present invention, the reaction conditions in step 1) are as follows: stirring the reaction at 70-95°C for 8-12h, for example, the reaction temperature is 70°C, 75°C, 80°C, 85°C, 90°C, etc., and the reaction time is, for example, 8h, 9h, 10h, 11h, 12h, etc.

[0020] As a preferred embodiment of the preparation method of the present invention, in step 1), solvent A is one or more of methanol, ethanol, butanol and propylene glycol.

[0021] As a preferred embodiment of the preparation method of the present invention, in step 2), the molar ratio of the intermediate product to sodium azide is 1:(1-1.5), for example, 1:1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, etc.

[0022] As a preferred embodiment of the preparation method of the present invention, the reaction conditions in step 1) are stirring the reaction at 90-120°C for 8-12h, for example, the reaction temperature is 90°C, 95°C, 100°C, 105°C, 110°C, 115°C, etc., and the reaction time is, for example, 8h, 9h, 10h, 11h, 12h, etc.

[0023] As a preferred embodiment of the preparation method of the present invention, in step 2), solvent B is a mixture of water and dimethylformamide, and the mixing mass ratio of the two is 1:(5-10), for example, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, etc.

[0024] As a preferred embodiment of the preparation method of the present invention, after the reaction in step 1) is completed, the reaction solution is cooled to room temperature and filtered to obtain a solid, which is the intermediate product;

[0025] Preferably, after the reaction in step 2) is completed, the reaction solution is poured into ice water while hot to obtain a solid which is then separated and purified by column chromatography to obtain the target product, the fluorescent probe.

[0026] A fluorescent probe for detecting H2S gas signal molecules as described above or a fluorescent probe for detecting H2S gas signal molecules prepared by the method described above is used in living cell fluorescence imaging.

[0027] The fluorescent probe provided by the present invention responds quickly to H2S at in vivo temperature, exhibits strong fluorescence, and has high detection sensitivity, and is particularly suitable as a fluorescent probe targeting endogenous H2S gas signal molecules. DETAILED DESCRIPTION

[0028] The present invention is further described below by means of specific examples. The examples described in the present invention are only used to illustrate the present invention and do not limit the scope of the present invention.

[0029] [Example 1]

[0030] 500 mg (1.8 mmol) of 4-bromo-1,8-naphthalene dicarboxylic anhydride and 500 mg (3.4 mmol) of 4-(2-aminoethyl)-thiomorpholine were dissolved in 100 mL of ethanol and stirred under reflux at 80° C. for 8 hours. After cooling to room temperature, the solid was filtered and dried in a vacuum oven to obtain an intermediate product (6.2 g, yield 89%).

[0031] 350mg (5.4mmol) of sodium azide was dissolved in 5mL of water, and the intermediate product (2.0g, 5.1mmol) was dissolved in 30mL of DMF. Then sodium azide was slowly added dropwise to the intermediate product solution, and all were mixed and stirred at 100°C for 10 hours. After the reaction was completed, the reaction solution was poured into ice water while hot, and solids were precipitated, filtered, and dried to obtain powder. The product was separated and purified by column chromatography (eluent was dichloromethane / methanol with a volume ratio of 50:1) to obtain the target product fluorescent probe.

[0032] 1 H NMR (300MHz, DMSO) δ8.51(d,J=12.0Hz,1H),8.50(d,J=8.0Hz,1H),8.49(d,J=8.0Hz,1H),8.25(d,J=8.0Hz,1H) ,7.87(t,J=8.0Hz,1H),3.16(t,J=6.0Hz,2H),2.73(t,J=4.0Hz,4H),2.57(t,J=6.0Hz,2H),2.54(J=4.0Hz,4H).

[0033] [Example 2]

[0034] 500 mg (1.8 mmol) of 4-bromo-1,8-naphthalene dicarboxylic anhydride and 292 mg (2 mmol) of 4-(2-aminoethyl)-thiomorpholine were dissolved in 100 mL of ethanol and stirred under reflux at 70° C. for 12 hours. After cooling to room temperature, the solid was filtered and dried in a vacuum oven to obtain an intermediate product.

[0035] 455mg (7mmol) of sodium azide was dissolved in 5mL of water, and the intermediate product (2.0g, 5.1mmol) was dissolved in 30mL of DMF. Then sodium azide was slowly added dropwise to the intermediate product solution, and all were mixed and stirred at 90°C for 12 hours. After the reaction was completed, the reaction solution was poured into ice water while hot, and solids were precipitated, filtered, and dried to obtain powder. The product was separated and purified by column chromatography (eluent was dichloromethane / methanol with a volume ratio of 50:1) to obtain the target product fluorescent probe.

[0036] [Example 3]

[0037] 500 mg (1.8 mmol) of 4-bromo-1,8-naphthalene dicarboxylic anhydride and 657 mg (4.5 mmol) of 4-(2-aminoethyl)-thiomorpholine were dissolved in 100 mL of ethanol and stirred under reflux at 95° C. for 10 hours. After cooling to room temperature, the solid was filtered and dried in a vacuum oven to obtain an intermediate product.

[0038] 390 mg (6 mmol) of sodium azide was dissolved in 5 mL of water, the intermediate product (2.0 g, 5.1 mmol) was dissolved in 30 mL of DMF, and sodium azide was slowly added dropwise to the intermediate product solution. After all were mixed, the mixture was stirred at 120 ° C for 8 hours. After the reaction was completed, the reaction solution was poured into ice water while hot, and the solid was precipitated, filtered, and dried to obtain a powder. The product was separated and purified by column chromatography (eluent was dichloromethane / methanol with a volume ratio of 50:1) to obtain the target product fluorescent probe.

[0039]

Application examples

[0040] (1) H2S response test of the probe:

[0041] First, a 1 mmol / L aqueous solution of the fluorescent probe prepared in Example 1 was prepared, and its absorption spectrum was tested at about 400 nm. Then, using NaHS as the H2S donor, a 1 μmol / L NaHS solution was added to the probe solution, and a sharp decrease in absorbance at 400 nm was detected, while a new absorption band appeared at a longer wavelength of 450 nm. When observed with the naked eye, the color of the solution changed from colorless to light yellow, indicating that visual detection of H2S can be achieved.

[0042] (2) Fluorescence intensity test at specific temperature:

[0043] 1 μmol / L NaHS solution was added to the prepared 1 mmol / L fluorescent probe aqueous solution, and the solution temperature was kept at 37°C. Under 365 nm ultraviolet light, the solution showed a gradually increasing fluorescence response within 30 minutes. It is particularly noteworthy that a high fluorescence intensity was detected within 5 minutes, indicating that the H2S detection sensitivity is high. The continuous detection results are shown in Table 1:

[0044] Table 1. Fluorescence intensity test results at specific temperatures

[0045] Time / min 0 5 10 15 20 25 30 35 40 Fluorescence intensity / au 5 53 128 180 228 266 278 280 283

[0046] Generally, the reaction speed of the fluorescent probe and H2S is affected by temperature. The higher the temperature, the faster the reaction. However, at the lower body temperature (37°C), the response speed of the fluorescent probe is limited to a certain extent. However, it can be seen from the test results in Table 1 that the fluorescent probe provided by the present invention can complete the H2S response relatively quickly (within 30 minutes) even at the body temperature, indicating that it is obviously more suitable for living cell fluorescence imaging and the application of endogenous H2S gas signal molecule targeted fluorescent probes in this respect.

[0047] (3) Detection sensitivity test:

[0048] The fluorescent probe solution (final concentration of 1 μmol / L) was added to NaHS solutions of different concentrations (final concentration of 5, 10, 25, 50, 100, 250 nmol / L) to react, and the fluorescence spectrum of the solution after the reaction was measured. The results showed that when reacting with different concentrations of NaHS (in the range of 5-250 nmol / L), the fluorescent probe produced a "turn on" type of fluorescence signal change, indicating that the fluorescence detection has high sensitivity and a wide range of adaptability.

[0049] (4) Fluorescence selectivity test:

[0050] The following common anion compounds in nature or the human body, including KCl, NaI, MgSO4, KNO3, NaNO2, Na2CO3, NaHCO3, NaOAc, and aqueous solutions (1 μmol / L) of common active small molecules, including glutathione, cysteine, glutamic acid, proline, serine, 5-aminolevulinic acid, and arginase, were added to 1 mmol / L aqueous solutions of the fluorescent probe respectively. The results showed that the fluorescence emission intensity of these solution samples did not change significantly compared with the fluorescence emission intensity of the fluorescent probe alone (Φ=0.03), indicating that the fluorescent probe is highly responsive and selective only to H2S.

[0051] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A fluorescent probe for detecting H2S gas signal molecules, characterized in that: The fluorescent probe has the following structural expression:

2. A method for preparing a fluorescent probe for detecting H2S gas signal molecules, characterized in that: The following steps are involved: 1) dissolving 4-bromo-1,8-naphthalene dicarboxylic anhydride and 4-(2-aminoethyl)-thiomorpholine in solvent A, stirring and reacting under reflux conditions to obtain an intermediate product; 2) The intermediate product and sodium azide are mixed in solvent B, and reacted by stirring under reflux conditions to generate the target product fluorescent probe.

3. The method for preparing a fluorescent probe for detecting H2S gas signal molecules according to claim 2, characterized in that: In step 1), the molar ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride to 4-(2-aminoethyl)-thiomorpholine is 1:(1-2.5).

4. The method for preparing a fluorescent probe for detecting H2S gas signal molecules according to claim 3, characterized in that: The reaction conditions in step 1) are stirring the reaction at 70-95° C. for 8-12 hours.

5. The method for preparing a fluorescent probe for detecting H2S gas signal molecules according to claim 3 or 4, characterized in that: In step 1), solvent A is one or more of methanol, ethanol, butanol and propylene glycol.

6. The method for preparing a fluorescent probe for detecting H2S gas signal molecules according to any one of claims 2 to 4, characterized in that: In step 2), the molar ratio of the intermediate product to sodium azide is 1:(1-1.5).

7. The method for preparing a fluorescent probe for detecting H2S gas signal molecules according to claim 6, characterized in that: The reaction conditions in step 1) are stirring the reaction at 90-120° C. for 8-12 hours.

8. The method for preparing a fluorescent probe for detecting H2S gas signal molecules according to claim 6, characterized in that: In step 2), solvent B is a mixture of water and dimethylformamide, and the mass ratio of the two is 1:(5-10).

9. The method for preparing a fluorescent probe for detecting H2S gas signal molecules according to any one of claims 2 to 4, characterized in that: After the reaction in step 1) is completed, the reaction solution is cooled to room temperature and filtered to obtain a solid, which is the intermediate product.

10. The method for preparing a fluorescent probe for detecting H2S gas signal molecules according to claim 9, characterized in that: After the reaction in step 2) is completed, the reaction solution is poured into ice water while hot to obtain a solid which is then separated and purified by column chromatography to obtain the target product, the fluorescent probe.

11. Use of the fluorescent probe for detecting H2S gas signal molecules as claimed in claim 1 or the fluorescent probe for detecting H2S gas signal molecules prepared by the method described in any one of claims 2 to 10 in living cell fluorescence imaging for non-disease diagnosis or treatment purposes.

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