Preparation and Application of a Near-Infrared Fluorescent Molecular Probe with "ESIPT + AIE" Effect

Abstract

A near-infrared fluorescent molecular probe with the "ESIPT + AIE" effect for detecting hydrogen sulfide has the following structural formula: #imgabs0# The aqueous solution of this fluorescent molecular probe is sensitive to the concentration of H2S, with a detection limit (LOD) of 0.83 μM (S / N = 3), showing high sensitivity; the maximum emission wavelength is 713 nm, having advantages such as strong tissue penetration, low background fluorescence interference, and little damage to cells / tissues, which is conducive to realizing the detection and / or imaging of endogenous H2S content; the linear detection range is 0 - 30 μmol / L, with a wider quantitative range and a broader application range.

Description

Technical Field

[0001] The present invention relates to the preparation and application of a fluorescent molecular probe, and particularly to the preparation and application of a near-infrared fluorescent molecular probe with an "ESIPT + AIE" effect for detecting hydrogen sulfide, belonging to the technical field of analytical detection. Background Art

[0002] Hydrogen sulfide (H 2 S) is the third gaseous signaling molecule found to have physiological regulatory functions after nitric oxide and carbon monoxide. It is reported that H 2 S participates in numerous physiological processes in organisms, including vasodilation and generation, apoptosis, tissue inflammation, and neuromodulation processes. At the same time, abnormal levels of H 2 S in organisms may lead to diseases such as Alzheimer's disease, diabetes, and Down syndrome. Therefore, developing effective tools to achieve the detection and (or) imaging of endogenous H 2 S content is of great significance.

[0003] Fluorescent probes are powerful tools for specifically detecting endogenous H 2 S in biological samples. Currently, various different types of organic fluorophores have been used for the fluorescent detection and (or) imaging of intracellular endogenous H 2 S with high spatiotemporal resolution. However, most common fluorescent probes for H 2 S detection will produce an aggregation-caused quenching (ACQ) effect in aqueous biological samples, resulting in fluorescence quenching. In contrast, some novel fluorophores with aggregation-induced emission (AIE) characteristics can not only overcome this defect but also have other good characteristics, such as higher brightness, lower background noise, and better photostability. At the same time, near-infrared fluorescent molecular probes have the advantages of strong tissue penetration, less background fluorescence interference, and less damage to cells / tissues, which are more conducive to achieving the detection and (or) imaging of endogenous H 2 S content.

[0004] For example, the invention patent with the application number CN201911322945.4 and the name "A preparation method and application of a hydrogen sulfide ratio-type fluorescent molecular probe based on the ESIPT effect" applied by the applicant before proposed a near-infrared molecular probe for detecting hydrogen sulfide, but its maximum emission wavelength is 608 nm, and it is still easily interfered by background fluorescence during cell imaging, and it is necessary to further extend its maximum emission wavelength.

[0005] There is also an invention patent with the application number CN202110273219.9 and the title "Preparation and Application of a Hydrogen Sulfide Fluorescent Probe Based on Isophorone-Xanthene", which proposes a hydrogen sulfide fluorescent probe with a large Stokes shift and near-infrared emission. However, its detection linear range is 1 μM - 10 μM, which needs to be improved.

[0006] For the invention patent with the application number CN202110248260.0 and the title "Preparation and Application of a 1,8-Naphthalimide-Based Hydrogen Sulfide Fluorescent Molecular Probe", a fluorescent molecular probe with a linear detection range width of 0 - 40 μM is proposed. However, its maximum emission wavelength is relatively short, and its maximum emission wavelength needs to be further extended. Summary of the Invention

[0007] In view of the problem that the current fluorescent molecular probes for detecting hydrogen sulfide cannot simultaneously achieve a long wavelength and a wide linear detection range, the present invention proposes the preparation and application of a near-infrared fluorescent molecular probe with an "ESIPT + AIE" effect for detecting hydrogen sulfide, achieving the effects of a long emission wavelength and a wide quantitative range at the same time.

[0008] The technical means adopted by the present invention to solve the above problems is: a near-infrared fluorescent molecular probe with an "ESIPT + AIE" effect for detecting hydrogen sulfide, having the structural formula shown in Formula I:

[0009]

[0010] A preparation method of a near-infrared fluorescent molecular probe with an "ESIPT + AIE" effect for detecting hydrogen sulfide, the steps are as follows:

[0011] (1) Dissolve 2-hydroxyacetophenone and terephthalaldehyde in an alcohol solvent such as ethanol, methanol, isopropanol, etc., add KOH, reflux and stir at 40 - 60 °C, monitor the reaction by TLC until it is complete, cool to room temperature, adjust the pH to near neutral with a 1 - 3 M hydrochloric acid solution, perform vacuum filtration, wash and dry the filter cake, and then purify by column chromatography to obtain a red solid compound I; the molar ratio of 2-hydroxyacetophenone, terephthalaldehyde, and KOH is 1:1:2.5 - 1:1.2:3, and the dosage ratio of 2-hydroxyacetophenone to the alcohol solvent is 1 mmol:6 mL - 1 mmol:10 mL;

[0012] (2) Dissolve Compound I in an alcohol solvent, add 1,4-dimethylpyridinium iodide salt and piperidine, reflux at 90 °C, monitor the reaction by TLC until completion, cool, and remove the solvent by rotary evaporation. Purify by column chromatography to obtain Compound II; the molar ratio of Compound I, 1,4-dimethylpyridinium iodide salt, and piperidine is 1:1.2:0.3 to 1:1.8:0.6, and the dosage ratio of Compound I to the alcohol solvent is 1 mmol: 3.5 mL to 1 mmol: 6 mL;

[0013] (3) Under the protection of nitrogen or argon, dissolve Compound II and 2,4-dinitrochlorobenzene in anhydrous acetonitrile, add K 2 CO 3 , stir and react at room temperature, monitor the reaction process by TLC. After the reaction is completed, concentrate under reduced pressure to remove acetonitrile to obtain a crude product, and purify by silica gel column chromatography to obtain the target molecular probe shown in Formula I; the molar ratio of Compound II, 2,4-dinitrochlorobenzene, and K 2 CO 3 is 1:1.2:1.2 to 1:2.4:2.4, and the dosage ratio of Compound II to anhydrous acetonitrile is 1 mmol: 12 mL to 1 mmol: 24 mL.

[0014] The reaction route is as follows:

[0015]

[0016] Application of a near-infrared fluorescent molecular probe with an "ESIPT + AIE" effect for detecting hydrogen sulfide, wherein the fluorescent molecular probe is used for quantitative detection of hydrogen sulfide in an aqueous system and imaging of endogenous hydrogen sulfide in living cells.

[0017] The specific steps for the near-infrared fluorescent molecular probe shown in Formula I to detect H 2 S are as follows:

[0018] (I) Add different amounts of Na 2 S solid to a PBS buffer solution (0.01 M, pH = 7.4) to prepare H 2 S solutions with different contents, and add the compound shown in Formula I with the same concentration to each of them to prepare at least five standard solutions containing the compound shown in Formula I with different H 2 S contents;

[0019] The concentration of the compound shown in Formula I in the standard solution is 1 μM to 20 μM;

[0020] The H 2 S content in the standard solution is 0.1 nM to 1 mM;

[0021] (2) Measure the fluorescence emission spectra of the standard solutions respectively, with an excitation wavelength of 560 nm. Using the H 2 S concentration as the abscissa and the fluorescence emission peak intensity value (I 713 nm ) at 713 nm as the ordinate, establish a standard curve;

[0022] (3) Add the compound shown in Formula I to the sample to be measured, and control its concentration to be equal to the concentration of the compound shown in Formula I in the standard solution; measure its fluorescence emission spectrum under the excitation light with an excitation wavelength of 560 nm, and then calculate the H 2 S content of the sample to be measured according to the standard curve.

[0023] The present invention constructs a classical ESIPT-coupled AIE system using 2-hydroxyacetophenone fluorophore, and introduces a strong electron-withdrawing group 4-methylpyridine at the 4'-position. On the one hand, it enhances the ICT effect of the probe, causing a large red shift in the maximum fluorescence emission wavelength to reach the near-infrared region. On the other hand, it also enhances the water solubility of the probe. Introducing 2,4-dinitrochlorobenzene on the hydroxyl group can quench the fluorescence through the photoinduced electron transfer (PET) effect, and at the same time block the generation of the ESIPT process. When the probe responds to H 2 S, free hydroxyl groups are generated, thus restoring the ESIPT effect and resulting in the emission of red fluorescence. The reaction principle is as follows:

[0024]

[0025] The beneficial effects of the present invention are:

[0026] 1. The fluorescent molecular probe provided by the present invention is a red solid powder with good water solubility and is convenient to use.

[0027] 2. The aqueous solution of the fluorescent molecular probe provided by the present invention is sensitive to the concentration of H 2 S, with a detection limit (LOD) of 0.83 μM (S / N = 3), high sensitivity. As the concentration of H 2 S increases, the red fluorescence gradually strengthens, and there is a good linear relationship, which can be used for the quantitative detection of H 2 S.

[0028] 3. The maximum emission wavelength of the fluorescent molecular probe provided by the present invention is 713 nm, which has the advantages of strong tissue penetration, small background fluorescence interference, and small damage to cells / tissues, etc., facilitating the detection and / or imaging of endogenous H 2 S content.

[0029] 4. The near-infrared fluorescent molecular probe with the "ESIPT + AIE" effect provided by the present invention has a linear detection range of 0 - 30 μmol / L, a wider quantitative range, and a wider application range. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Emission spectrum of the fluorescence molecular probe prepared in the implementation of the present invention with the fluorescence intensity varying with the concentration of H 2 S;

[0031] Figure 2 Linear relationship between the fluorescence molecular probe prepared in the implementation of the present invention and the concentration of H 2 S;

[0032] Figure 3 Selectivity diagram of the fluorescence molecular probe prepared in the implementation of the present invention for H 2 S;

[0033] Figure 4 Fluorescence imaging diagram of the fluorescence molecular probe prepared in the implementation of the present invention for intracellular endogenous H 2 S. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention will be further described below with reference to the accompanying drawings.

[0035] Example 1

[0036] Synthesis of Compound I

[0037] 2-Hydroxyacetophenone (408.45 mg, 3 mmol) and terephthalaldehyde (402.40 mg, 3 mmol) were added to a 50 mL round-bottom flask. After adding 25 mL of ethanol to completely dissolve them, KOH (420.75 mg, 7.5 mmol) was added to the reaction system. The reaction system was refluxed and stirred at 60 °C, and monitored by TLC until the reaction was complete. The reaction system was cooled to room temperature, and its pH was adjusted to near neutral with 3 M hydrochloric acid solution. A large amount of precipitate was visible. After vacuum filtration, washing and drying the filter cake, it was separated and purified by silica gel column chromatography to obtain 615.28 mg of a red solid, with a yield of 81.3%. 1 H NMR (400 MHz, CDCl 3 ): δ 13.36 (s, 1H), 10.23 (s, 1H), 7.98 (dd, J = 8.3, 6.9 Hz, 2H), 7.63 (d, J = 8.9 Hz, 2H), 7.54 - 7.47 (m, 2H), 7.08 (dd, J = 8.3, 0.8 Hz, 1H), 6.99 - 6.94 (m, 2H), 6.79 (d, J = 8.9 Hz, 2H).

[0038] Synthesis of Compound II

[0039] Add compound I (605.45 mg, 2.4 mmol) and 10 mL of ethanol to a 25 mL round-bottom flask. After complete dissolution, add 1,4-dimethylpyridinium iodide (842.4 mg, 3.6 mmol) and 1 mmol of piperidine, and reflux at 90 °C. Monitor the reaction by TLC until completion. After cooling, remove the solvent by rotary evaporation and purify by column chromatography to obtain 602.61 mg of the product with a yield of 53.5%. 1 HNMR(400MHz,CDCl 3 ): δ 14.87 (s, 1H), 8.12 (d, J = 8.4, 2H), 8.08 (d, J = 8.9 Hz, 1H), 7.94 (d, J = 8.2, 2H), 7.64 - 7.57 (m, 2H), 7.47 - 7.45 (m, 2H), 7.31 (d, J = 8.0, 4H), 6.96 (d, J = 8.2 Hz, 1H), 6.95 - 6.90 (m, 2H), 4.40 (s, 3H).

[0040] Synthesis of the fluorescent molecular probe shown in Formula I

[0041] Under argon protection, add compound II (469.32 mg, 1 mmol) and 2,4-dinitrochlorobenzene (303.84 mg, 1.5 mmol) to a 25 mL round-bottom flask. Add 20 mL of anhydrous acetonitrile to dissolve them completely, and add K 2 CO 3 (207.3 mg, 1.5 mmol) to the reaction system. Stir the reaction at room temperature and monitor the reaction progress by TLC. After the reaction is completed, concentrate under reduced pressure to remove acetonitrile to obtain the crude product, and purify by silica gel column chromatography to obtain 395.84 mg of the probe with a yield of 62.3%. 1 H NMR(400MHz,CDCl 3 ): δ 8.88 (s, 1H), 8.83 (d, J = 8.2, 2H), 8.41 (d, J = 8.0, 1H) 8.22 (d, J = 8.0, 2H), 8.08 - 8.06 (m, 2H), 7.73 (t, J = 7.9, 1H), 7.61 (d, J = 6.8, 1H), 7.47 (t, J = 7.6, 1H), 7.32 (d, J = 7.8, 1H), 7.29 (d, J = 7.8, 4H), 7.10 (d, J = 8.0, 1H), 6.94 - 6.91 (m, 2H), 4.38 (s, 3H). HRMS-ESI (m / z) calc. for C 29 H 22 IN 3 O 6 [M - I] +: 508.15031; found 508.23194.

[0042] Example 2

[0043] Fluorescent molecular probe for H 2 Fluorescence response experiment of S

[0044] Prepare a PBS buffer solution with a concentration of 10 mM and a pH of 7.4 as reagent stock solution a. Dissolve the fluorescent probe shown in Formula I prepared in Example 1 in a PBS buffer solution (0.01 M, pH = 7.4) to prepare a solution with a concentration of 1 mM as reagent stock solution b. The volume ratio of reagent stock solution a and reagent stock solution b in the reagent kit is 200:1.

[0045] Dissolve the fluorescent probe solution b (1 mM, 20 μL) in reagent stock solution a (10 mM, 1960 μL), and then add 20 μL of Na 2 S standard solution (aqueous solution) with concentrations of 0 μM, 0.5 mM, 1 mM, 2 mM, 3 mM, 5 mM, 7.5 mM, 10 mM, 15 mM, and 20 mM. The Na 2 S standard solution is prepared by Na 2 S stock solution (concentration of 1 M) and PBS buffer solution (0.01 M, pH = 7.4) such that in the detection system, the final concentration of the fluorescent molecular probe is 10 μM, while the final concentration of H 2 S is 0 μM, 5 μM, 10 μM, 20 μM, 30 μM, 50 μM, 75 μM, 100 μM, 150 μM, and 200 μM. Incubate at room temperature for 20 min and measure its fluorescence spectrum. The fluorescence emission spectrum change diagram is shown in Figure 1 . The results show that as the concentration of H 2 S increases, the fluorescence intensity of the system gradually increases at 713 nm. Figure 2 Is the linear diagram of the fluorescence intensity change of the fluorescent molecular probe prepared in Example 1 at 713 nm versus the concentration of H 2 S. It can be obtained from Figure 2 that there is a good linear relationship in the range of 0 - 30 μM. The linear equation is y = 4.22307x + 4.54476, and the linear correlation coefficient is: 0.99544. The detection limit (LOD) is calculated to be 0.83 μM (S / N = 3), indicating that the fluorescent probe has good sensitivity.

[0046] Example 3

[0047] Fluorescent molecular probe for H 2 Selectivity experiment of S

[0048] Dissolve the fluorescent probe solution b (1 mM, 20 μL) in the reagent stock solution a (10 mM, 1960 μL), and then add 20 μL of Na 2 S standard solution and metal ions (Na + , K + , Mg 2+ ), anions (F - , Cl - , Br - ), and the mother liquor of biomacromolecules (Cys, Hcy, GSH), so that the final concentration of the probe in the entire detection system is 10 μM, while the concentration of H 2 S is 10 μM, and the concentrations of metal ions (Na + , K + , Mg 2+ ), anions (F - , Cl - , Br - ), and biomacromolecules (Cys, Hcy, GSH) are all 30 μM. Then perform fluorescence scanning and calculate the relative fluorescence intensity in each system; use the relative fluorescence intensity (I 713 nm ) at 713 nm as the ordinate to obtain the response bar chart of the probe to different substances (see Figure 3 ). It can be seen that the fluorescent probe only responds to H 2 S and has excellent anti-interference ability.

[0049] Example 4

[0050] Imaging application of the fluorescent molecular probe to endogenous H 2 S in living cells

[0051] Place two portions of HepG2 cells in a medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics, and culture them at 37 °C for 48 h in a humid environment containing 5% CO 2 . Use a microinjector to aspirate the reagent stock solution b (diluted from the 1 mM reagent stock solution b with the reagent stock solution a) into the medium containing HepG2 cells to make the probe concentration 10 μM, and continue to culture in the incubator for 30 min, and perform fluorescence imaging. As can be seen from Figure 4 a), obvious red fluorescence can be seen, indicating that the fluorescent molecular probe or detection kit of the present invention can directly detect endogenous H 2 S in cells. In order to verify that the fluorescence signal in the cells indeed comes from the response of the fluorescent molecular probe to H 2 S. The control group uses H 2The cells were pretreated with the S scavenger NMM for 30 min, and then the reagent stock solution b (diluted from 1 mM reagent stock solution b with reagent stock solution a) was added and incubated for another 30 min before fluorescence imaging. As Figure 4 shown in b) of 2 , almost no fluorescence was observed in the cells, indicating that the fluorescence signal in the cells indeed originated from the response of the fluorescent molecular probe or the detection kit to endogenous H

[0052] S in the cells. Since near-infrared (NIR) fluorescent probes have the advantages of low background interference, strong tissue penetration, and little tissue damage, and the window with the maximum emission wavelength in the range of 700 - 1700 nm has gradually been proven to be an optical "transparent" window for biological tissues. When it penetrates biological tissues such as skin, fat, and bone, there are fewer scattering and absorption phenomena, so the "attenuation rate" is lower. Moreover, when the maximum emission light is greater than 700 nm, the autofluorescence from various pigments in the living body is also greatly reduced. With the help of these two major advantages, fluorescence imaging in this region has good performance and great development prospects in cell or in vivo animal imaging research. In addition, when the fluorescence intensity of the fluorescent probe has a larger linear range with the H 2 S concentration, the linear range of H 2 S that this fluorescent probe can detect is wider. The maximum emission wavelength of the fluorescent molecular probe in this example reaches 713 nm, and the linear detection range reaches 0 - 30 μmol / L, and it also has the advantages of strong anti-interference ability and wide detection range.

[0053] The above embodiments are only for illustrating the present invention, rather than limiting the present invention. Those skilled in the relevant technical fields can also make various modifications and changes without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions should also fall within the protection scope of the present invention, and the protection scope of the present invention should be defined by each claim.

Claims

1. A near-infrared fluorescent molecular probe with the "ESIPT + AIE" effect for detecting hydrogen sulfide, Characterized in that: It has the structural formula shown in Formula I:

2. A preparation method of the near-infrared fluorescent molecular probe with the "ESIPT + AIE" effect for detecting hydrogen sulfide according to claim 1, Characterized in that: The preparation steps are as follows: (1) Dissolve 2-hydroxyacetophenone and terephthalaldehyde in an alcohol solvent, add KOH, heat under reflux, cool to room temperature after the reaction ends, adjust the pH to near neutral, filter under reduced pressure, wash and dry the filter cake, and then purify by column chromatography to obtain a red solid compound I, where the structural formula of compound I is: (2) Dissolve compound I in an alcohol solvent, add 1,4-dimethylpyridinium iodide salt and piperidine, heat under reflux until the reaction ends, cool, concentrate under reduced pressure, and purify by column chromatography to obtain compound II, where the structural formula of compound II is: (3) Under the protection of inert gas, dissolve compound II and 2,4-dinitrochlorobenzene in anhydrous acetonitrile, add K 2 CO 3 . After the stirring reaction is completed at room temperature, concentrate under reduced pressure and purify by silica gel column chromatography to obtain the target molecular probe shown in formula I.

3. The preparation method of the near-infrared fluorescent molecular probe with the "ESIPT + AIE" effect for detecting hydrogen sulfide according to claim 2, Characterized in that: The alcohol solvent is ethanol, methanol, or isopropanol.

4. The preparation method of the near-infrared fluorescent molecular probe with the "ESIPT + AIE" effect for detecting hydrogen sulfide according to claim 2, Characterized in that: The reflux temperature in step (1) is 40 - 60 °C.

5. The preparation method of the near-infrared fluorescent molecular probe with the "ESIPT + AIE" effect for detecting hydrogen sulfide according to claim 2, Characterized in that: In step (1), 1 - 3M hydrochloric acid solution is used to adjust the pH to near neutral; the molar ratio of 2-hydroxyacetophenone, terephthalaldehyde, and KOH is 1:1:2.5 - 1:1.2:3, and the dosage ratio of 2-hydroxyacetophenone to the alcohol solvent is 1 mmol:6 mL - 1 mmol:10 mL.

6. The preparation method of the near-infrared fluorescent molecular probe with the "ESIPT + AIE" effect for detecting hydrogen sulfide according to claim 2, Characterized in that: The reflux temperature in step (2) is 90 °C.

7. The preparation method of the near-infrared fluorescent molecular probe with the "ESIPT + AIE" effect for detecting hydrogen sulfide according to claim 2, Characterized in that: In step (2), the molar ratio of compound I, 1,4-dimethylpyridinium iodide salt, and piperidine is 1:1.2:0.3 - 1:1.8:0.6, and the dosage ratio of compound I to the alcohol solvent is 1 mmol:3.5 mL - 1 mmol:6 mL.

8. The preparation method of the near-infrared fluorescent molecular probe with the "ESIPT + AIE" effect for detecting hydrogen sulfide according to claim 2, Characterized in that: In step (3), the molar ratio of compound II, 2,4-dinitrochlorobenzene, and K 2 CO 3 is 1:1.2:1.2 to 1:2.4:2.4, and the dosage ratio of compound II to anhydrous acetonitrile is 1 mmol:12 mL to 1 mmol:24 mL.

9. An application of the near-infrared fluorescent molecular probe with the "ESIPT + AIE" effect for detecting hydrogen sulfide according to claim 1, Characterized in that: The fluorescent molecular probe is used for the quantitative detection of hydrogen sulfide in an aqueous system.

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