A carbazole fluorescent probe for rapid and efficient detection of H2S, its synthesis method and application

Abstract

The present invention belongs to the field of fluorescent probe technology, specifically a carbazole fluorescent probe for rapid and efficient detection of H2S, its synthesis method, and application. The compound is 9-(4-(2,4-dinitrophenoxy)phenyl)-9H-carbazole, having a structure of formula (1): #imgabs0#. The probe can selectively identify H2S molecules. The fluorescent probe uses a carbazole molecule as a fluorescent group and then links a 2,4-dinitrophenyl group at the position of the hydroxyl group to form a carbazole fluorescent compound. The carbazole fluorescent probe is a fluorescent probe based on a thiolysis reaction. In the presence of H2S, a thiolysis reaction can occur rapidly, removing the 2,4-dinitrophenyl group and emitting strong fluorescence. It can be widely used to detect H2S in cells, soil, and water.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and specifically relates to a carbazole fluorescent probe for rapid and efficient detection of H2S, a synthesis method thereof, and an application thereof. Background Art

[0002] Hydrogen sulfide (H2S) is a gas with the smell of rotten eggs. It often appears in heavy industrial processes such as metal smelting and coal mining, as well as in the long-term decay and fermentation of organic matter. In mild cases, it can cause sensory discomfort and irritation of the nervous system. In severe cases, it can cause poisoning or even rapid death.

[0003] As a signaling molecule, H2S participates in several important physiological processes, including blood pressure regulation, neurotransmission, and suppression of oxidative stress. Excessive H2S levels are associated with a variety of diseases, including Down syndrome, Parkinson's disease, and diabetes. H2S is an active molecule that regulates biological systems, but abnormal levels are closely linked to dysfunction and numerous diseases. This has spurred the development of small molecule chemical tools to investigate the diverse roles of H2S in biology and medicine. Beyond its lethal effects, H2S also possesses several beneficial properties, mediating a wide range of physiological actions, including vasodilation, anti-inflammation, insulin release, neurotransmission, antioxidant activity, anti-apoptosis, and neuroprotection. For example, H2S induces sulfation of Keap1, leading to Nrf2 activation and nuclear translocation, and the synthesis of antioxidant proteins. Numerous studies have shown that H2S is the third most important signaling molecule in organisms, after CO and NO. H2S has been detected in a growing number of fields. To gain a more detailed understanding of its role in physiological processes, it is crucial to track its specific changes within complex environments and physiological processes.

[0004] Electrochemical methods based on sulfide-selective electrodes, methylene blue assays, colorimetry, spectrophotometry, high-performance liquid chromatography, and gas chromatography, among others, are often limited by time, labor, and testing costs, making them inefficient for the qualitative and quantitative analysis of H2S in cells. Therefore, to accurately and reliably measure H2S concentrations in aqueous, gaseous, and living cells (both exogenous and endogenous), a simple and rapid method for detecting hydrogen sulfide in cell tissues is required. Summary of the Invention

[0005] The present invention addresses the shortcomings of the prior art by providing a carbazole fluorescent probe for rapid and efficient H2S detection and a synthesis method thereof. This probe can selectively recognize H2S molecules. The fluorescent probe uses a carbazole molecule as a fluorescent group, then links a phenol at the nitrogen-hydrogen bond position as a linker, and then links a 2,4-dinitrophenyl chloride to the hydroxyl group of the phenol to form a carbazole fluorescent compound. The 2,4-dinitrophenyl chloride not only inhibits the ICT process in the carbazole fluorophore but also serves as an H2S recognition site. The carbazole fluorescent probe is based on a thiolysis reaction. In the presence of H2S, the ether bond is attacked, rapidly undergoing thiolysis, removing the 2,4-dinitrophenyl group and emitting strong fluorescence. The probe can be widely used for H2S detection within cells, soil, and water.

[0006] In order to achieve the above object of the invention, the specific technical solution of the present invention is:

[0007] A carbazole fluorescent probe compound for rapid and efficient detection of H2S, the compound being 9-(4-(2,4-dinitrophenoxy)phenyl)-9H-carbazole (abbreviated as fluorescent probe ZST-107), has the structure of formula (1):

[0008] The compound is used for rapid and efficient detection of H2S.

[0009] A method for preparing a carbazole fluorescent probe compound for rapid and efficient detection of H2S, the synthesis route of which is shown in formula (2):

[0010]

[0011] A method for preparing a carbazole fluorescent probe compound for rapid and efficient detection of H2S comprises the following steps:

[0012] (1) Copper powder, 18-Crown-6, and K2CO3 were added to a mixture of Compound A and Compound B, which was then dissolved in DMSO. The mixture was refluxed under argon and detected by TLC. After the reaction was complete, the mixture was extracted with ethyl acetate and recrystallized to obtain colorless crystals of Compound C.

[0013] (2) Under argon and ice bath conditions, compound C was dissolved in dichloromethane, and BBr3 was slowly added dropwise. The reaction temperature was gradually raised to room temperature and detected by TLC. After the reaction was complete, the mixture was extracted with dichloromethane and dried in a rotary evaporator to obtain a gray solid compound D.

[0014] (3) K2CO3 was added to the mixture of Compound D and Compound E, and the mixture was dissolved in DMF. The mixture was stirred at a certain temperature and detected by TLC. After the reaction was completed, the yellow solid Compound F was obtained by recrystallization.

[0015] As a better embodiment of the present application, in step (1) of the method for preparing a rapid and efficient detection of H2S carbazole fluorescent probe compound, the molar ratio of compound A, compound B, copper powder, 18-Crown-6, and K2CO3 is 1:1-1.5.0:5-10:0.3-1.0:5.0-10.0, and each millimole of compound A is dissolved in 5-15 mL of DMSO. The reaction conditions are 150±10°C and 8±0.5 h.

[0016] As a better embodiment of the present application, in step (2) of the preparation method of a rapid and efficient detection of H2S carbazole fluorescent probe compound, the molar ratio of compound C and BBr3 is 1:1.2~5.0; each millimole of compound C is dissolved in 5~10mL of dichloromethane, and the reaction conditions are 0±3℃, 3±0.5h.

[0017] As a better embodiment of the present application, in step (3) of the preparation method of a rapid and efficient detection of H2S carbazole fluorescent probe compound, the molar ratio of compound D, compound E and potassium carbonate is 1:1.0-2.0:1.5-3.0, each millimole of compound C is dissolved in 5-15 mL of DMF, and the reaction conditions are 100±5°C and 8±0.5h.

[0018] The present application also protects the use of the aforementioned carbazole fluorescent probe compound for rapid and efficient detection of H2S in cells, soil or water.

[0019] Furthermore, the reaction mechanism of its application is shown in formula (3);

[0020]

[0021] The present invention uses carbazole molecules as fluorescent groups. After the fluorescent groups are introduced into 2,4-dinitrophenyl, the final structure is obtained. The two nitro groups on the introduced 2,4-dinitrophenyl have a strong electron-withdrawing effect, so that the fluorescence emitted by the carbazole fluorescent group is quenched. The fluorescent probe undergoes a sulfur decomposition reaction with H2S to remove the 2,4-dinitrophenyl group, the fluorescence quenching effect disappears, and the fluorescence effect at 371.4nm is significantly enhanced.

[0022] In specific applications, the specific operation of qualitative detection can be referred to as follows: add the sample to be tested into the solution containing the probe ZST-107, mix evenly, and observe its color change under a 365nm ultraviolet lamp. If the probe emits strong orange-yellow fluorescence, it contains hydrogen sulfide.

[0023] The present invention also specifically protects the use of a carbazole fluorescent probe in the quantitative detection of H2S in an aqueous environment.

[0024] In specific applications, quantitative detection can refer to the following specific operations: add the sample to be tested to a solution containing the probe ZST-107, mix evenly, and perform fluorescence spectrophotometry detection at an excitation wavelength of 293 nm and an emission wavelength range of 340 nm to 450 nm. Within a certain range, the linear equation of fluorescence intensity y and H2S concentration x is: y = (3304.0379 ± 284.98446) * x + (768.80408 ± 292.88999).

[0025] The linear equation for the quantitative detection of fluorescence intensity y and H2S concentration x has a high degree of fitting, R 2 =0.952.

[0026] The carbazole fluorescent probe of the present invention exhibits strong signal enhancement capabilities in detecting hydrogen sulfide in aquatic environments, with fluorescence enhancement of up to 108.24 times at an emission wavelength of 371.4 nm. It enables qualitative analysis of hydrogen sulfide with the naked eye, with a detection limit of 66 nM, demonstrating the ability to monitor low levels of hydrogen sulfide. Furthermore, monitoring of hydrogen sulfide in various natural water environments demonstrates its strong anti-interference capability, with a good linear relationship between the fluorescence intensity of ZST-107 and the concentration of hydrogen sulfide.

[0027] The present invention also specifically protects the use of a carbazole fluorescent probe in the quantitative detection of endogenous H2S in living cells.

[0028] In specific applications, quantitative detection can refer to the following specific operations:

[0029] The cytotoxicity of the probe ZST-107 was studied using A549 cells (lung tumor cells) and CCK-8 reagent. After treatment with 20 μM ZST-107 for 12 hours, 95% of the A549 cells remained viable. These results demonstrate that the probe exhibits low cytotoxicity to A549 cells and can be used to detect endogenous H2S in living cells.

[0030] A549 cells were first incubated with ZST-107 (10 μM) for 30 min, and then Cys (cysteine) was added and incubated together. We observed green fluorescence in the cells.

[0031] Experimental results show that the carbazole fluorescent probe ZST-107 can be used to detect endogenous H2S in living cells.

[0032] Compared with the existing technology, the beneficial effects of the present invention are:

[0033] (1) The preparation method of the carbazole fluorescent probe compound is simple and easy and has a high overall yield.

[0034] (2) The carbazole fluorescent probe compound is widely used in H2S detection in cells, soil and water.

[0035] (3) The carbazole fluorescent probe compound has low toxicity to cells and can be used to detect endogenous H2S in living cells.

[0036] (IV) The carbazole fluorescent probe compound has high sensitivity, and the detection limit of the probe is calculated to be 66 nM.

[0037] (5) The carbazole fluorescent probe compound has an extremely fast response speed, and its complete detection time only takes 60 seconds. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The synthetic route of the fluorescent probe ZST-107 of the present invention is shown in FIG.

[0039] Figure 2 This is the ultraviolet concentration gradient spectrum of the fluorescent probe ZST-107 of the present invention;

[0040] Figure 3 This is the concentration titration diagram of H2S under fluorescence conditions of the fluorescent probe ZST-107 of the present invention;

[0041] Figure 4 This is a test chart of the selectivity of the fluorescent probe ZST-107 of the present invention to H2S;

[0042] Figure 5 This is a test chart of the anti-interference performance of the fluorescent probe ZST-107 of the present invention against H2S;

[0043] Figure 6 This is the time response diagram of the fluorescent probe ZST-107 of the present invention in recognizing H2S;

[0044] Figure 7 This is the titration diagram of H2S concentration of the fluorescent probe ZST-107 under ultraviolet conditions;

[0045] Figure 8 This is a diagram of the cytotoxicity test of the fluorescent probe ZST-107 of the present invention.

[0046] Figure 9 This is the hydrogen spectrum of the nuclear magnetic resonance characterization of the fluorescent probe ZST-107 of the present invention. DETAILED DESCRIPTION

[0047] All features disclosed in this specification, or all steps in the disclosed methods or processes, except mutually exclusive features and / or steps, can be combined in any manner.

[0048] Any feature disclosed in this specification (including claims and abstract), unless otherwise stated, may be replaced by other equivalent or similar features. That is, unless otherwise stated, each feature is only an example of a series of equivalent or similar features.

[0049] The features and properties of the present invention are further described in detail below with reference to the examples. % in the following examples, unless otherwise specified, represents volume percentage; steps not described in detail are conventional techniques.

[0050] Example 1:

[0051] A fast and efficient detection of H2S carbazole fluorescent probe compound, the compound is 9-(4-(2,4-dinitrophenoxy)phenyl)-9H-carbazole, and its structural formula is:

[0052]

[0053] Example 2:

[0054] A fast and efficient detection of H2S carbazole fluorescent probe compound, the preparation route of which is as follows:

[0055]

[0056] The specific method includes the following steps:

[0057] Preparation of compound C:

[0058]

[0059] To a mixture of compound A (1.67 g, 10 mmol, 1 eq) and compound B (3.51 g, 15 mmol, 1.5 eq) were added copper powder (4.9 g, 75 mmol, 7.5 eq), 18-Crown-6 (1.32 g, 5 mmol, 0.5 eq), and K2CO3 (10.37 g, 75 mmol, 7.5 eq). The mixture was then dissolved in 40 mL of DMSO and refluxed at 150°C overnight under argon. The reaction was monitored by TLC. After completion of the reaction, the mixture was extracted with ethyl acetate and recrystallized from DCM / PE. This afforded compound C (2.5 g, 91.5%) as colorless crystals.

[0060] Preparation of compound D:

[0061]

[0062] Under argon and an ice bath, compound C (7.5 g, 27 mmol, 1 eq) was dissolved in 100 mL of dichloromethane, and BBr (8.1 g, 32.4 mmol, 1.2 eq) was slowly added dropwise. The temperature was gradually raised to room temperature for 3 h. The reaction was monitored by TLC. After completion, the mixture was extracted with dichloromethane and dried to dryness. This afforded compound D (6.9 g, 98.67%) as a gray solid.

[0063] Preparation of compound F:

[0064]

[0065] To a mixture of compound D (420.5 mg, 1.62 mmol, 1 eq) and compound E (394.5 mg, 1.95 mmol, 1.2 eq) was added KCO (336 mg, 2.43 mmol, 1.5 eq) and dissolved in 15 mL of DMF. The mixture was stirred at 100°C for 8 h and analyzed by TLC. After completion of the reaction, compound F (i.e., probe molecule ZST-107) was recrystallized from methanol and ethyl acetate to yield a yellow solid (526 mg, 76.33%). The overall yield for this route was 68.9%.

[0066] Example 3:

[0067] Detection characteristics and performance of carbazole H2S fluorescent probe.

[0068] 1. Spectral property test of probe molecule ZST-107

[0069] The fluorescent probe prepared in Example 2 (Compound F, concentration of 10.0 μM, solvent is 20% (mass percentage) DMSO in PBS) was used to detect hydrogen sulfide (0-30eq sodium hydrosulfide, solvent is water), with an excitation wavelength of 293 nm and an emission wavelength of 371.4 nm. The results are as follows: Figure 2 shown.

[0070] Depend on Figure 3 It can be seen that the fluorescent probe has a very weak emission peak at 371.4nm. After adding excess H2S, the fluorescence enhancement at 371.4nm is significantly enhanced and increases with the increase of hydrogen sulfide concentration, up to 108 times the original. When 3 times the amount of H2S is added, the fluorescence intensity basically reaches saturation, indicating that the fluorescent probe can realize the detection of H2S in solution. The calculated detection limit is 66nM.

[0071] 2. Test of the selectivity of optical probe ZST-107 for H2S:

[0072] 100 μM analyte (K + , Cu + , Ca 2+ , Na + , I - , F - , Cl - , CO3 2- ,HCO3 - , S2O8 2- , HSO4 - , HSO3 - , SCN - , S2O3 2- , S2O5 2- , SO3 2- , NO2 - , L-Cys, D-Cys, GSH, Zinc Citrate), and the change of fluorescence intensity of the detection solution at 371.4nm. Figure 4 As shown in the figure, ZST-107 exhibits an obvious fluorescence response to H2S, while the increase in fluorescence intensity is limited and almost negligible when coexisting with other analytes, indicating that the fluorescent probe ZST-107 has good selectivity for recognizing H2S.

[0073] 3. Test of the anti-interference ability of fluorescent probe ZST-107 to H2S:

[0074] 100 μM analyte (K + , Cu + , Ca 2+ , Na + , I - , F - , Cl - , CO3 2- ,HCO3 - , S2O8 2- , HSO4 - , HSO3 - , SCN - , S2O3 2- , S2O5 2- , SO3 2- , NO2 -, L-Cys, D-Cys, GSH, Zinc Citrate), and then 100 μM H2S was added to the mixed solution containing other analytes, and the fluorescence emission spectrum at 371.4 nm was detected. Figure 5 As shown in the figure, when the above-mentioned interfering analytes coexist, ZST-107 is still effective for H2S. It can be seen that the presence of other ions does not affect the recognition performance of the fluorescent probe ZST-107 for H2S, and it has good anti-interference ability.

[0075] 4. Test of the response time of fluorescent probe ZST-107 to H2S:

[0076] To a 10 μmol / L PBS buffer solution of the fluorescent probe ZST-107 (pH=7.4, containing 20% ​​DMSO and 100 μM CTAB), 100 μM H2S was added and the fluorescence intensity was measured every 30 seconds. Figure 6 The fluorescence intensity gradually increased with time and reached saturation around 60 s, indicating that the recognition of H2S by probe ZST-107 could be completed in around 60 s. The fluorescence intensity increased linearly from 0 to 60 s, indicating that the probe had a fast response capability.

[0077] 5. Cytotoxicity test of fluorescent probe ZST-107:

[0078] The cytotoxicity of the probe ZST-107 was studied using A549 cells (lung tumor cells) and CCK-8 reagent. A549 cells (lung tumor cells) were seeded in a 96-well plate and incubated for 24 hours (37°C, 5% CO2) before detection. The old culture medium was removed, and the probe ZST-101 (0μM, 3μM, 5μM, 10μM, 20μM, 30μM) with the same volume and different concentrations was added and continued to incubate for 12 hours under the above conditions. The original culture medium was aspirated and then gently washed three times with PBS buffer. CCK solution (0.500mg / mL, 100μL) was then added to each well. After culturing for 4 hours, the absorbance at 372nm was measured with an enzyme reader to calculate the cytotoxicity. Figure 8 As shown, 95% of A549 cells remained viable after being treated with 20 μM ZST-107 for 12 hours. The experimental results indicate that the probe exhibits low toxicity to A549 cells and can be used to detect endogenous H2S in living cells.

[0079] The above-described embodiments merely represent specific implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the present application. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the technical concept of the present application, and all such variations and improvements fall within the scope of protection of the present application.

[0080] This background section is provided to generally present the context of the invention, and the work of the presently named inventors, the work to the extent described in this background section, and aspects of the description in this section that did not constitute prior art at the time of filing are neither explicitly nor implicitly admitted to be prior art to the present invention.

Claims

1. A carbazole fluorescent probe compound for rapid and efficient detection of H2S, characterized in that: The compound is 9-(4-(2,4-dinitrophenoxy)phenyl)-9H-carbazole, having the structure of formula (1): (1), the compound is used to detect H2S.

2. Use of the carbazole fluorescent probe compound for rapid and efficient detection of H2S as claimed in claim 1 in rapid and efficient detection of H2S in soil or water.

3. The use according to claim 2, characterized in that The mechanism of this application is recorded as formula (3); （3）。 4. The use according to claim 2, characterized in that The test includes qualitative and quantitative testing. In qualitative testing, the sample to be tested is added to a solution containing a carbazole fluorescent probe compound, and the color change is observed under a 365nm ultraviolet lamp. If the probe emits strong orange-yellow fluorescence, it contains hydrogen sulfide.

5. The use according to claim 2, characterized in that During quantitative detection, the sample to be tested was added to a solution containing a carbazole fluorescent probe compound, and fluorescence spectrophotometry was performed at an excitation wavelength of 293 nm and an emission wavelength range of 340 nm to 450 nm. Within a certain range, the linear equation between the fluorescence intensity y and the H2S concentration x was: y = (3304.0379 ± 284.98446) * x + (768.80408 ± 292.88999).

6. A method for preparing a carbazole fluorescent probe compound for rapid and efficient detection of H2S, characterized in that: Its synthetic route is shown in formula (2): （2）。 7. The method for preparing a carbazole fluorescent probe compound for rapid and efficient detection of H2S according to claim 6, characterized in that The following steps are involved: (1) Copper powder, 18-Crown-6, and K2CO3 were added to the mixture of compound A and compound B, mixed evenly, dissolved in DMSO, and refluxed under argon; detected by TLC, extracted with ethyl acetate after the reaction was complete, and recrystallized to obtain colorless crystal compound C; (2) Under argon and ice bath conditions, compound C was dissolved in dichloromethane, and BBr3 was slowly added dropwise thereto. The reaction temperature was gradually raised to room temperature and detected by TLC. After the reaction was complete, the compound was extracted with dichloromethane and dried to obtain a gray solid compound D. (3) Add K2CO3 to the mixture of compound D and compound E, and dissolve it in DMF; stir at a certain temperature, detect by TLC, and recrystallize to obtain yellow solid compound F after the reaction is completed.

8. The method for preparing a carbazole fluorescent probe compound for rapid and efficient detection of H2S according to claim 7, characterized in that: In step (1), the molar ratio of compound A, compound B, copper powder, 18-Crown-6, and K2CO3 is 1:1-1.5.0:5-10:0.3-1.0:5.0-10.0; 5-15 mL of DMSO is used to dissolve each millimole of compound A, the reaction temperature is 150±10°C, and the reaction time is 8±0.5 h.

9. The method for preparing a carbazole fluorescent probe compound for rapid and efficient detection of H2S according to claim 7, characterized in that: In step (2), the molar ratio of compound C to BBr3 is 1:1.2-5.0; each millimole of compound C is dissolved in 5-10 mL of dichloromethane, and the reaction conditions are 0±3°C and 3±0.5 h.

10. The method for preparing a carbazole fluorescent probe compound for rapid and efficient detection of H2S according to claim 7, characterized in that: In step (3), the molar ratio of compound D, compound E and potassium carbonate is 1:1.0-2.0:1.5-3.0, and each millimole of compound G is dissolved in 5-15 mL of DMF. The reaction conditions are 100±5°C and 8±0.5h.

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