Novel quinoline H2S fluorescent probe and preparation method thereof

Abstract

The invention relates to the technical field of fluorescence detection, in particular to a novel quinoline H2S fluorescent probe and a preparation method thereof. The novel quinoline H2S fluorescent probe is designed and synthesized by adopting a reaction strategy of reducing nitroso into amino and taking a quinoline derivative as a fluorophore. The aminoquinoline fluorophore has good optical performance, raw materials involved in synthesis are cheap, and the method has the advantages of being simple in reaction, mild in reaction condition and the like. After the probe reacts with hydrogen sulfide, nitroso groups are reduced into hydroxylamine groups and amino groups, the fluorescence of the compound is enhanced, and effective detection of H2S can be realized.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence detection technology, and in particular relates to a novel quinoline H2S fluorescent probe and its preparation method. Background Technology

[0002] Hydrogen sulfide (H2S) is an important endogenous gaseous signaling molecule involved in regulating various physiological activities, including immunity, the nervous system, the cardiovascular system, and the endocrine system. Under physiological pH conditions, H2S is mainly expressed as HS-H2S. - H2S exists in various forms, and its homeostasis is crucial for maintaining normal function. Abnormal concentrations have been proven to be closely related to the occurrence and development of various diseases, including Alzheimer's disease, diabetes, and cirrhosis. Therefore, developing technologies capable of efficiently detecting H2S in living cells, tissues, and organisms is of significant scientific value for elucidating its mechanisms of action in physiological and pathological processes.

[0003] To detect H2S, various methods have been developed, including colorimetric methods, electrochemical analysis, chromatography, and metal sulfide precipitation. However, these traditional methods typically have inherent limitations, such as insufficient sensitivity, complex sample pretreatment, and difficulty in achieving real-time in-situ cell monitoring, which greatly restricts their application in complex biological systems. Fluorescent probe methods, due to their advantages of high sensitivity, good selectivity, high resolution, and minimal invasiveness to biological samples, have become important tools for detecting trace amounts of bioactive molecules in vivo. Based on this, the design of fluorescent probes for H2S has been extensively studied. Currently, reported H2S fluorescent probes are mainly based on two reaction mechanisms: nucleophilic reactions and reduction reactions. Nucleophilic probe design strategies include the thiolysis of aromatic nitro groups, the exchange of disulfide (selenium) bonds, nucleophilic addition to unsaturated bonds, and tandem Michael addition. Probes based on the reducing properties of H2S typically utilize the reduction of azides, nitro groups, and hydroxylamines to amino groups, thereby triggering significant changes in fluorescence signals and achieving detection.

[0004] Despite their diverse mechanisms, existing probes still face challenges in terms of performance and application. In particular, reports on H2S fluorescent probes based on the novel reaction pathway of nitrosyl reduction to amino groups are still very limited. For example, Chen et al. recently reported an H2S probe using benzopyran dye as the fluorophore and nitrosyl as the recognition group. However, such probes generally suffer from insufficient sensitivity and unsatisfactory selectivity, making it difficult to meet the needs of high-precision biological detection. Furthermore, the synthetic routes of most probes are complex and cumbersome, hindering large-scale preparation and practical application. Therefore, developing a simple, fast-responding, highly sensitive, and selective H2S fluorescent probe, especially a high-performance probe based on the nitrosyl reduction mechanism, is of urgent practical significance for advancing H2S-related biological research and disease diagnosis. Summary of the Invention

[0005] The technical problem this invention aims to solve is to address the shortcomings of the prior art by providing a novel quinoline H2S fluorescent probe and its preparation method. Employing a reaction strategy of reducing nitrosyl groups to amino groups, and using quinoline derivatives as fluorescent groups, a novel quinoline H2S fluorescent probe was designed and synthesized. This probe has a simple synthetic route, a short response time to H2S, and a certain degree of anti-interference capability, making the detection process faster and more reliable, and thus suitable for H2S detection applications.

[0006] This invention provides a novel quinoline H2S fluorescent probe, the structural formula of which is: .

[0007] This invention also provides a method for preparing the above-mentioned novel quinoline H2S fluorescent probe, comprising the following steps:

[0008] S1. Take 4,4,4-trifluoro-1-(p-tolyl)butane-1,3-dione and m-phenylenediamine, add chloroform to dissolve, react in an oil bath with stirring under reflux, concentrate by rotary evaporation, then recrystallize and wash, filter and dry to obtain the intermediate product.

[0009] S2. Mix the intermediate product with dichloromethane to obtain a mixed solution. Dissolve sodium persulfate in distilled water to obtain an aqueous solution of potassium persulfate. Add the potassium persulfate aqueous solution dropwise to the mixed solution and stir the reaction at room temperature. After the reaction is complete, extract three times with an extractant. Separate and combine the resulting lower organic layers, dry and filter them, concentrate by rotary evaporation, and then purify by column chromatography to obtain a novel quinoline H2S fluorescent probe.

[0010] According to the preparation method provided by the present invention, the ratio of the amount of 4,4,4-trifluoro-1-(p-tolyl)butane-1,3-dione, m-phenylenediamine and chloroform in S1 is 1.00 g: 0.50-1.00 g: 15-25 mL.

[0011] According to the preparation method provided by the present invention, the temperature of the oil bath in S1 is 80 °C, the stirring and reflux reaction time is 12 h, the solvent for recrystallization is chloroform, and the washing solution is n-hexane.

[0012] According to the preparation method provided by the present invention, the ratio of the intermediate product, dichloromethane, sodium persulfate and distilled water in S2 is 0.5 mmol: 5-15 mL: 2.5-5.0 mmol: 10-20 mL.

[0013] According to the preparation method provided by the present invention, the stirring reaction time in S2 is 24 h, the stirring speed is 1000 r / min, the extractant is dichloromethane, the drying agent is anhydrous Na2SO4, and the eluent for column chromatography is petroleum ether:ethyl acetate = 2:1.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] This invention provides a novel quinoline H2S fluorescent probe and its preparation method. A novel quinoline H2S fluorescent probe is designed and synthesized by using a reaction strategy of reducing nitroso to amino groups and using quinoline derivatives as fluorescent groups.

[0016] The probe has a simple synthesis route, a short response time to H2S, and a certain degree of anti-interference capability, making the detection process faster and more reliable, and can be put into H2S detection applications. Attached Figure Description

[0017] Figure 1 The graph shows the reaction detection of probe 4 with different concentrations of NaHS.

[0018] Figure 2 The graph shows the fluorescence intensity of probe 4 reacting with NaHS over time (a) and the fluorescence intensity of the probe after the addition of different interfering ions (b).

[0019] Figure 3 The mass spectrum of the reaction between probe 4 and NaHS;

[0020] Figure 4 For the HOMO and LUMO molecular orbitals of compounds 3, 4 and 5;

[0021] Figure 5 The diagram shows the surface electrostatic potentials of compounds 3, 4, and 5. Detailed Implementation

[0022] In all the following examples, common chemicals and solvents (analytical grade) were purchased from commercial suppliers and did not require purification. Distilled water was used in all examples. 200-300 mesh silica gel for column chromatography was purchased from Qingdao Ocean. The UV-2550 spectrophotometer was purchased from Shimadzu, Japan. The FL-4600 fluorescence spectrophotometer was purchased from Shimadzu, Japan. The Bruker Avance-600 nuclear magnetic resonance spectrometer was purchased from Bruker, Germany.

[0023] Example 1

[0024] This embodiment provides a method for preparing a novel quinoline H2S fluorescent probe. The reaction process structure is as follows: ;

[0025] Includes the following steps: S1. Take a 50 mL round-bottom flask and add 4,4,4-trifluoro-1-(p-tolyl)butane-1,3-dione (1.00 g, 4.63 mmol) and m-phenylenediamine (0.50 g, 4.63 mmol) to the flask in sequence, and add 15 mL of chloroform. Then immerse the flask in an oil bath at 80 °C to carry out the reaction. The system presents a transparent yellow-green solution. Stir and reflux for 12 h. After the reaction is completed by TLC with PE:EA = 10:1 as the developing solvent, the solid is concentrated by rotary evaporation and then recrystallized with chloroform, washed with n-hexane, filtered and dried to obtain aminoquinoline compound 3, which is a light green fluorescent needle-like crystal. The yield of compound 3 was 0.9410 g, with a yield of 68%. 1 H NMR (600 MHz, CDCl3) δ(ppm) 8.04 (d, J = 8.0 Hz, 2H), 7.93 - 7.91(m, 1H), 7.86 (s, 1H), 7.35 - 7.33 (m, 3H), 7.06 - 7.04 (m, 1H), 4.16 (s,2H), 2.43 (s, H); MS (ESI) m / z: calcd for C 17 H 14 F3N 2+ 303.30 [M+H] + ;found:303.11; S2. Take a 50 mL round-bottom flask, add compound 3 (0.15 g, 0.5 mmol) to the flask, and add 5 mL of dichloromethane to dissolve it. Dissolve sodium persulfate (4.20 g, 2.5 mmol) in 10 mL of distilled water to obtain an aqueous solution of potassium persulfate. Add the potassium persulfate aqueous solution dropwise to the round-bottom flask and stir vigorously at room temperature for 24 h at a stirring speed of 1000 r / min. After the reaction is complete, extract three times with dichloromethane. Separate and combine the lower organic layers, dry with anhydrous Na2SO4 and filter to obtain a clear yellow-green liquid. After rotary evaporation and concentration, the crude product is purified by column chromatography with PE:EA = 2:1 (petroleum ether: ethyl acetate) as the eluent, and probe 4 (i.e., compound 4) is an orange-red solid. MS (ESI) m / z: calcd for C 17 H 14 F3N 2+ 317.28 [M+H] + ; found: 317.30.

[0026] Example 2

[0027] This embodiment provides a method for preparing a novel quinoline H2S fluorescent probe.

[0028] Includes the following steps: S1. Take a 50 mL round-bottom flask, add 1.0 g of 4,4,4-trifluoro-1-(p-tolyl)butane-1,3-dione and 1.0 g of m-phenylenediamine to the flask in sequence, and add 25 mL of chloroform. Then immerse the flask in an oil bath at 80 °C to carry out the reaction. The system presents a transparent yellow-green solution. Stir and reflux for 12 h. After the reaction is completed by TLC with PE:EA = 10:1 as the developing solvent, the solid is concentrated by rotary evaporation to obtain a yellow-green solid. Then recrystallize with chloroform, wash with n-hexane, filter and dry to obtain aminoquinoline compound 3, which is a light green fluorescent needle-like crystal. S2. Take a 50 mL round-bottom flask, add 0.5 mmol of compound 3 to the flask, and simultaneously add 15 mL of dichloromethane to dissolve it. Dissolve 5.0 mmol of sodium persulfate in 20 mL of distilled water to obtain an aqueous solution of potassium persulfate. Add the potassium persulfate aqueous solution dropwise to the round-bottom flask, and stir vigorously at room temperature for 24 h at a stirring speed of 1000 r / min. After the reaction is complete, extract three times with dichloromethane, separate and combine the lower organic layers, dry with anhydrous Na2SO4 and filter to obtain a clear yellow-green liquid. After rotary evaporation and concentration, the crude product is purified by column chromatography with PE:EA = 2:1 as the eluent, and probe 4 is obtained as an orange-red solid.

[0029] Example 3 The fluorescence detection method for hydrogen sulfide using probe 4 prepared in Example 1: The synthesized 3.00 mg probe 4 was dissolved in 10.00 mL of dimethyl sulfoxide (DMSO) solution to obtain a 1 mmol / L stock solution; During optical measurements, different concentrations of probe 4, 0.5 mL of phosphate buffer, and different concentrations of NaHS are added to a colorimetric tube, diluted with water to 5.0 mL, and the test solution is obtained. Then, its absorption spectrum or fluorescence spectrum is measured.

[0030] The parameters of the fluorescence spectrophotometer are set as follows: λ Ex / Em =290 / 420 nm, E X and E m The slit width is 5.0 nm.

[0031] The maximum emission peak of aminoquinoline compound 3 was determined to be 540 nm using a fluorescence spectrophotometer. 500 μL of phosphate buffer solution (pH 7.4) and different doses of NaHS solution were added to colorimetric tubes containing 5 μM probe 4. Figure 1 As shown in (a), with the continuous addition of NaHS, the absorbance of the system increases with the increase of NaHS concentration (λ=320nm), accompanied by a slight red shift.

[0032] At the same time, such as Figure 1 As shown in (b), the fluorescence of the reaction system at 370 nm is significantly enhanced and there is a noticeable red shift, indicating the formation of new substances.

[0033] The effect of pH on the optical properties of probe 4 was further investigated. Figure 1 As shown, the fluorescence intensity of probe 4 did not change significantly within the pH range of 6.0–8.0, indicating that probe 4 is stable within the physiological pH range; with the addition of NaHS, the fluorescence intensity gradually increased; since aromatic nitroso compounds are similar to other nucleophiles, HS... - Nucleophiles attack the positively charged N in N=O, thereby rapidly reacting to generate hydroxylamine intermediates or directly generating amino compounds.

[0034] Under different pH conditions, such as Figure 1 As shown in (d), when pH=8.0, the reaction rate of probe 4 with NaHS is faster, and the peak value gradually increases with increasing concentration. A large amount of hydroxylamine compounds are generated in a short time, and more aminoquinoline derivatives are generated at 540 nm, such as... Figure 1 (d) As shown in the inset diagram.

[0035] Conversely, Figure 1 As shown in (c), at pH=6.0, probe 4 does not react with NaHS at all.

[0036] Example 4

[0037] 100 μM NaHS was added to a 5 μM probe solution, and the kinetics of the response of probe 4 to hydrogen sulfide were studied by detecting the change in fluorescence intensity at 370 nm.

[0038] like Figure 2 As shown in (a), at pH=8.0, the fluorescence intensity of the reaction system significantly increases with time. The reaction is relatively rapid in the first 10 minutes, but the fluorescence intensity remains essentially unchanged after 50 minutes, indicating that probe 4 has good sensitivity to H2S.

[0039] Further investigation into the selectivity of probe 4, such as Figure 2As shown in (b), after adding each interfering ion, the fluorescence intensity of the reaction system at 370 nm increased slightly, but remained essentially unchanged.

[0040] Taking cysteine ​​(Cys) as an example, the fluorescence intensity only increased by 2.2 after adding 200 μM cysteine ​​to the reaction. However, when probe 4 reacted with NaHS, the fluorescence was significantly enhanced, indicating that the probe has good selectivity and other ions or small biomolecules do not seriously interfere with the detection of NaHS.

[0041] Example 5

[0042] Mechanism of probe 4 in recognizing hydrogen sulfide.

[0043] After probe 4 reacts with NaHS, its organic layer supernatant is analyzed by mass spectrometry, such as... Figure 3 As shown, the mass spectra after the reaction of the two compounds show two main molecular ion peaks at m / z 304.25870 and m / z 318.29785. Among them, the ion peak at m / z 304.25870 is aminoquinoline compound 3, and the other ion peak at m / z 318.29785 is hydroxylamine compound 5.

[0044] The structural formula of hydroxylamine compound 5 is: ;

[0045] Therefore, it can be concluded that after the probe 4 reacts with NaHS, it is reduced to aminoquinoline compound 3, but the product contains a large amount of hydroxylamine compound 5.

[0046] The aminoquinoline fluorophore possesses excellent optical properties, and its synthesis involves inexpensive raw materials and boasts advantages such as simple reaction and mild reaction conditions. After the probe reacts with hydrogen sulfide, the nitroso group is reduced to a hydroxylamine group and an amino group, resulting in enhanced fluorescence of the compound and enabling effective detection of H2S.

[0047] The mechanism of probe identification and detection of hydrogen sulfide is as follows: .

[0048] In addition, the structure and reactivity of the compound were analyzed using Gauss 09 software at the DFT / B3LYP / 6-311+G(d,p) level.

[0049] The structure was optimized using the B3LYP / 6-311+G(d,p) method, and the leading molecular orbitals and molecular surface electrostatic potentials were calculated. The results were plotted using Multiwfn and VMD.

[0050] like Figure 4As shown, the HOMO-LUMO band gaps of aminoquinoline compound 3, probe 4, and hydroxylamine compound 5 are 3.135 eV, 4.026 eV, and 4.107 eV, respectively. After the reaction of probe 4 with H2S, the electron clouds of the HOMO and LUMO orbitals of compounds 3 and 5 are located in the quinoline backbone, and the molecular structure plane changes, with the benzene ring rotating. The products after the probe is reduced by H2S exhibit better stability, with the hydroxylamine compound being the most stable.

[0051] Further investigation was conducted into the nucleophilic and electrophilic properties of the molecule.

[0052] like Figure 5 As shown, the calculated molecular surface electrostatic potential diagram presents the magnitude of the electrostatic potential in the surface region and is represented by different colors. The red region in the diagram has a negative electrostatic potential value, indicating that this region is more likely to donate electrons or is more nucleophilic than other regions; while the blue region has a positive electrostatic potential value, indicating that this region is more likely to gain electrons and is more electrophilic than other regions.

[0053] Depend on Figure 5 HS can be further verified - Nucleophiles attack the positively charged N in N=O, which makes the structure tend to be stable.

[0054] In summary, this invention designed and synthesized an H2S fluorescent probe with quinoline as the fluorophore and nitroso as the recognition group. By measuring the response of probe 4 to NaHS under different pH conditions, it was found that probe 4 and NaHS... - The active ingredient is reduced to hydroxylamine compounds and aminoquinoline derivatives, and the fluorescence intensity of the probe gradually increases, thereby achieving the purpose of detecting H2S.

[0055] At pH 8.0, probe 4 reacts with NaHS more rapidly, producing more amino compounds. Conversely, at pH 6.0, the probe hardly reacts with NaHS.

[0056] Mass spectrometry and DFT theory studies have verified the correctness of the recognition mechanism.

[0057] In addition, the probe has a simple synthesis route, a short response time to H2S, and a certain degree of anti-interference capability, making the detection process faster and more reliable, and it can be put into H2S detection applications.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A novel quinoline H2S fluorescent probe, characterized in that, The structural formula of the novel quinoline H2S fluorescent probe is: 。 2. A method for preparing the novel quinoline H2S fluorescent probe as described in claim 1, characterized in that, Includes the following steps: S1. Take 4,4,4-trifluoro-1-(p-tolyl)butane-1,3-dione and m-phenylenediamine, add chloroform to dissolve, react in an oil bath with stirring under reflux, concentrate by rotary evaporation, then recrystallize and wash, filter and dry to obtain the intermediate product. S2. Mix the intermediate product with dichloromethane to obtain a mixed solution. Dissolve sodium persulfate in distilled water to obtain an aqueous solution of potassium persulfate. Add the potassium persulfate aqueous solution dropwise to the mixed solution and stir the reaction at room temperature. After the reaction is complete, extract three times with an extractant. Separate and combine the resulting lower organic layers, dry and filter them, concentrate by rotary evaporation, and then purify by column chromatography to obtain a novel quinoline H2S fluorescent probe.

3. The preparation method according to claim 2, characterized in that, The ratio of 4,4,4-trifluoro-1-(p-tolyl)butane-1,3-dione, m-phenylenediamine, and chloroform in S1 is 1.00 g: 0.50–1.00 g: 15–25 mL.

4. The preparation method according to claim 2, characterized in that, The oil bath temperature in S1 is 80 °C, the stirring and reflux reaction time is 12 h, the solvent for recrystallization is chloroform, and the washing solution is n-hexane.

5. The preparation method according to claim 2, characterized in that, The ratio of the intermediate product, dichloromethane, sodium persulfate, and distilled water in S2 is 0.5 mmol: 5–15 mL: 2.5–5.0 mmol: 10–20 mL.

6. The preparation method according to claim 2, characterized in that, The stirring reaction time in S2 is 24 h, the stirring speed is 1000 r / min, the extractant is dichloromethane, the drying agent is anhydrous Na2SO4, and the eluent for column chromatography is petroleum ether:ethyl acetate = 2:1.

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