Preparation and application of water-soluble nitric oxide fluorescent probe based on cyanine dye

Abstract

The invention relates to preparation and application of a water-soluble nitric oxide fluorescent probe based on a cyanine dye. The structural formula of the fluorescent probe is # imgabs0. The invention provides a preparation method for synthesizing the fluorescent probe by taking the cyanine fluorescent dye, 1-adamantane formyl chloride, 3-dimethylamino propylamine and the like as raw materials. The fluorescent probe is a nitric oxide fluorescent probe with water solubility. Firstly, the fluorescent probe shows very high sensitivity to NO, and fluorescence is remarkably enhanced after the probe reacts with NO; secondly, the fluorescent probe shows good selectivity on NO and is not interfered by other common inorganic ions, active oxygen, active nitrogen and biological mercaptan; in addition, the fluorescent probe has been successfully used for NO imaging in cells, and can detect the nitric oxide level in the cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and particularly relates to the preparation and application of a water-soluble nitric oxide fluorescent probe based on cyanine dyes. Technical Background

[0002] Nitric oxide (NO), as a key signaling molecule, plays a crucial role in many physiological and pathological processes (S. Moncada, E. A. Higgs, Endogenous nitric oxide: physiology, pathology and clinical relevance, Eur. J. Clin. Chem., 1991, 21, 361 - 374). NO is endogenously produced by nitric oxide synthase (NOS) and participates in various physiological processes such as signal transduction, vasodilation, and smooth muscle relaxation (A. de Mel, F. Murad, A. M. Seifalian, Nitric oxide: a guardian for vascular grafts, Chem. Rev., 2011, 111, 5742 - 5767). The dysregulation of NO is associated with the generation of reactive nitrogen species, which are linked to some pathological processes, including cancer, inflammation, endothelial dysfunction, and neurodegeneration (A. G. Tennyson, S. J. Lippard, Generation, translocation, and action of nitric oxide in living systems, Chem. Biol., 2011, 18, 1211 - 1220). Therefore, developing effective methods to monitor NO in biological systems is of great value.

[0003] To date, many methods for detecting NO have been developed, including: colorimetric analysis (X.Q. Chen, F. Wang, J.Y. Hyun, T. Wei, J. Qiang, X. Ren, I. Shin, J. Yoon, Recent progress in the development of fluorescent, luminescent and colorimetric probes for detection of reactive oxygen and nitrogen species, Chem. Soc. Rev., 2016, 45, 2976-3016), electrochemical analysis (M.M. Musameh, C.J. Dunn, M.H. Uddin, T.D. Sutherland, T.D. Rapson, Silk provides a new avenue for third generation biosensors: sensitive, selective and stable electrochemical detection of nitric oxide, Biosens. Bioelectron., 2018, 103, 26-31), electron paramagnetic resonance spectroscopy (R. Rodriguez-Rodriguez, U. Simonsen, Measurement of nitric oxide and reactive oxygen species in the vascular wall, Curr. Anal. Chem., 2012, 8, 485-494) and chemiluminescence technology (Y.Y. Woldman, T.D. Eubank, A.J. Mock, N.C. Stevens, S. Varadharaj, J. Turco, M.A. Gavrilin, B.R. Branchini, V.V. Khramtsov, Detection of nitric oxide production in cell cultures by luciferin-luciferase chemiluminescence, Biochem. Biophys. Res. Commun., 2015, 465, 232-238). Compared with these traditional methods, fluorescence analysis methods have advantages such as high sensitivity and high spatio-temporal resolution.So far, many fluorescent probes for detecting NO have been reported (Z-P. Zheng, S-Y. Gong, J-Z. Zhang, Y-J. Liu, G-Q. Feng, Golgi-targeted fluorescent probe for nitric oxide imaging in Parkinson’s disease models, Sensor Actuat. B, 2023, 397, 134654; Z-L. Xu, S-T. Liu, L-R. Xu, Z-C. Li, X-L. Zhang, H. Kang, Y-F. Liu, J. Yu, J. Jing, G-L. Niu, X-L. Zhang, A novel ratiometric fluorescent probe with high selectivity for lysosomal nitric oxide imaging, Anal. Chim. Acta, 2024, 1297, 343303). However, these NO probes have some deficiencies: the analysis wavelength is relatively short and is easily interfered by the autofluorescence signals generated by biomolecules in vivo; the tissue penetration ability is weak, thus limiting their application in organisms. Therefore, it is very meaningful to design and synthesize NO fluorescent probes with longer analysis wavelengths.

[0004] Cyanine dyes are a type of dyes widely used in the field of fluorescent probes at present, with advantages such as large molar extinction coefficients and high photostability. Most importantly, they have near-infrared emission properties. Near-infrared emission can penetrate deeper tissues, is not easily interfered by autofluorescence of organisms, and is more beneficial for bioimaging. However, the poor water solubility of cyanine dyes limits their use in biological systems. Introducing adamantane onto cyanine dyes and utilizing its binding with β-cyclodextrin is expected to improve the water solubility of the dyes. Therefore, it is very necessary to design and synthesize a water-soluble fluorescent probe based on cyanine dyes as an effective tool for detecting NO in biological samples. Summary of the Invention

[0005] According to the requirements put forward, the inventors have conducted in-depth research on this. After a large amount of creative work, a water-soluble nitric oxide fluorescent probe based on cyanine dyes is provided.

[0006] The technical solution of the present invention is a water-soluble nitric oxide fluorescent probe based on cyanine dyes, namely Cy-Ac-NO, and its structure is as follows:

[0007]

[0008] A preparation method of a water-soluble nitric oxide fluorescent probe based on cyanine dye, and the reaction steps are as follows:

[0009] Dissolve 1 equivalent of Cy-Ac-Cl in 15 - 25 mL of anhydrous acetonitrile, add it to a 50 mL round-bottom flask, slowly add 4 - 6 equivalents of triethylamine and 4 - 6 equivalents of 3-dimethylaminopropylamine, and stir at 60 - 65 °C for 4 - 6 h under nitrogen protection; after the reaction is completed, the crude product is de-solventized under reduced pressure and purified with an eluent of CH2Cl2 / CH3OH with a volume ratio of 25:1 - 20:1 to obtain a blue solid compound Cy-Ac-NO, which is the fluorescent probe described above. Among them, the structure of Cy-Ac-Cl is as follows:

[0010]

[0011] The beneficial effect of the present invention is the good spectral response performance of a water-soluble nitric oxide fluorescent probe based on cyanine dye. First, study the fluorescence spectral properties of the probe. The fluorescent probe itself does not have an obvious near-infrared emission peak; after adding NO, an obvious near-infrared emission peak appears at 830 nm. And as the concentration of NO increases, the near-infrared fluorescence intensity of the probe continuously increases. When the NO concentration is 70 μM, the fluorescence intensity increases by 14.7 times. The detection range of this probe is from 3 μM to 70 μM, and the detection limit is 1 μM, indicating that this fluorescent probe can sensitively detect NO in the solution. Then, study the ultraviolet absorption spectrum of the probe. The probe does not show an obvious absorption band at 790 nm. After adding NO, the absorption peak that appears near 790 nm gradually increases, and the solution color changes from blue to light green. Then, study the selectivity of the probe. The probe was investigated with inorganic ions (K + , Ca 2+ , Na + , Mg 2+ , Ag + , Cu 2+ , Fe 3+ , NH4 + , Zn 2+ , CO3 2- , SO4 2- , Cl - , OH - ), reactive oxygen species (H2O2, ClO - ), reactive nitrogen species (NO2 - , ONOO -) And the fluorescence responses to biological thiols (Cys, Hcy, GSH). It was found that only NO could cause changes in the fluorescence spectrum, and other analytes had no obvious effect on the fluorescence spectrum of the probe. Finally, the effect of pH on the determination of NO by the fluorescence probe was studied. As the pH value increased, the fluorescence at 830 nm gradually enhanced. In the physiological environment of pH 7.4, the probe had a good response to NO, indicating that the probe had good prospects for application in biological samples.

[0012] Application of a water-soluble nitric oxide fluorescence probe based on cyanine dye. Macrophages without probe staining did not emit fluorescence; when the fluorescence probe Cy-Ac-NO was added to the cells, the fluorescence increased significantly; the cells were treated with lipopolysaccharide (LPS: an intracellular NO inducer), and then stained with the probe Cy-Ac-NO for imaging, and strong fluorescence was produced; after the cells were treated with lipopolysaccharide and then treated with N-nitro-L-arginine (L-NNA: an intracellular NO inhibitor), and finally stained with the probe Cy-Ac-NO for imaging, weak fluorescence was produced. These results indicate that the probe Cy-Ac-NO can sensitively detect NO in cells and provide a reliable means for monitoring NO-related lesions in biological samples. Description of the Drawings

[0013] Figure 1 is the synthetic route of the fluorescence probe.

[0014] Figure 2 is the fluorescence spectrum of the fluorescence probe after interacting with different concentrations of NO.

[0015] The abscissa is the wavelength and the ordinate is the fluorescence intensity. The concentration of the fluorescence probe is 10 μM, and the NO concentrations are: 0, 3, 5, 7, 10, 20, 30, 40, 50, 60, 70 μM. The fluorescence excitation wavelength is 790 nm.

[0016] Figure 3 is the fluorescence linear response diagram of the fluorescence probe to different NO concentrations.

[0017] Figure 4 is the UV-visible absorption spectrum of the fluorescence probe before and after interacting with NO.

[0018] The concentration of the fluorescence probe is 10 μM, and the NO concentration is 70 μM.

[0019] Figure 5 is the selectivity diagram of the fluorescence probe.

[0020] The concentration of the fluorescence probe is 10 μM, the NO concentration is 70 μM, and the concentrations of other analytes are all 70 μM.

[0021] Figure 6 is the diagram of the effect of pH on the fluorescence probe.

[0022] The concentration of the fluorescent probe is 10 μM, and the concentration of NO is 70 μM.

[0023] Figure 7 It is for the determination of the response time of the interaction between the fluorescent probe and NO.

[0024] The concentration of the fluorescent probe is 10 μM, and the concentrations of NO are 0, 10, 40, 70 μM.

[0025] Figure 8 It is for the cytotoxicity test.

[0026] The abscissa is the concentration of the fluorescent probe, and the ordinate is the cell survival rate.

[0027] Figure 9 Cell imaging diagram of the interaction between the fluorescent probe and NO.

[0028] Control group: Cells were stained with the probe Cy-Ac-NO for 30 min; LPS group: Cells were first treated with lipopolysaccharide (LPS) for 12 h, and then stained with the probe Cy-Ac-NO for 30 min; LPS+L-NNA group: Cells were first treated with lipopolysaccharide (LPS) and N-nitro-L-arginine (L-NNA) for 12 h, and then stained with the probe Cy-Ac-NO for 30 min.

[0029] Figure 10 It is the cell relative fluorescence intensity diagram. Detailed implementation manners

[0030] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but is not limited thereto.

[0031] Example 1:

[0032] Synthesis of the fluorescent probe

[0033] The synthesis route is as Figure 1 . Synthesis of the NO fluorescent probe (Cy-Ac-NO): Dissolve Cy-Ac-Cl (350 mg, 0.5 mmol) in 25 mL of anhydrous acetonitrile, add it to a 50 mL round-bottom flask, slowly add triethylamine (1.0 mL, 1.0 mmol) and 3-dimethylaminopropylamine (2.5 mL, 2.5 mmol), and stir at 65 °C for 6 h under nitrogen protection; after the reaction is completed, the crude product is de-solventized under reduced pressure and purified with a CH2Cl2 / CH3OH eluent with a volume ratio of 20:1 to obtain a blue solid compound (yield 65%), which is the fluorescent probe Cy-Ac-NO. 11H NMR (400 MHz, CDCl3) δ 7.50 - 7.55 (m, 1H), 7.33 - 7.38 (m, 1H), 7.21 - 7.29 (m, 6H), 7.01 - 7.08 (m, 3H), 6.92 (d, J = 8 Hz, 1H), 6.84 (d, J = 8 Hz, 1H), 5.56 (d, J = 12 Hz, 1H), 3.86 - 3.92 (m, 4H), 3.43 - 3.46 (m, 2H), 2.73 - 2.76 (t, J = 12 Hz, 2H), 2.56 (s, 2H), 2.48 - 2.51 (m, 2H), 2.08 (t, J = 12 Hz, 2H), 2.01 (s, 6H), 1.90 (d, J = 4.0 Hz, 6H), 1.82 (t, J = 12 Hz, 2H), 1.68 - 1.71 (m, 6H), 1.62 - 1.65 (s, 6H), 1.41 (s, 1H), 1.27 (s, 6H), 1.24 (s, 6H), 0.86 (t, J = 16 Hz, 3H). MS (TOF): 769.15.

[0034] Example 2:

[0035] Preparation of Fluorescent Probe and NO Solution

[0036] Preparation of probe solution: Weigh a certain amount of probe and β - cyclodextrin, and dissolve them in dimethyl sulfoxide (DMSO) in a molar ratio of 1:1 to prepare a 1×10 -4 M probe solution. Preparation of NO stock solution: Dissolve a certain amount of DEA·NONOate in secondary distilled water to prepare a 1×10 -3 M NO solution. Add 1 mL of the probe solution and different volumes of the NO stock solution to a 10 - mL volumetric flask, and make up the volume with PBS buffer solution at pH 7.4. Obtain the test solution containing 1.0×10 -5 M fluorescent probe and 1.0×10 -6 ~7.0×10 -5 M NO.

[0037] Example 3:

[0038] Determination of Fluorescence Spectrum of the Interaction between Fluorescent Probe and NO

[0039] Figure 2This is the fluorescence spectrum of the fluorescence probe interacting with NO. The concentration of the fluorescence probe is 10 μM, and the NO concentrations are 0, 3, 5, 7, 10, 20, 30, 40, 50, 60, 70 μM respectively. The fluorescence excitation wavelength is 790 nm, and the emission wavelength range is 800 - 900 nm. The excitation slit width is 5 nm, and the emission slit width is 10 nm. The fluorescence measurement instrument used is the Hitachi F4600 fluorescence spectrophotometer. It can be seen from the figure that due to the quenching effect of the 3-dimethylaminopropylamine group, the fluorescence probe itself does not have an obvious near-infrared emission peak; after adding NO, an obvious near-infrared emission peak appears at 830 nm. This is because NO binds to the 3-dimethylaminopropylamine group of the fluorescence probe, hindering electron transfer, thus generating near-infrared fluorescence. Moreover, as the NO concentration increases, the near-infrared fluorescence intensity of the probe molecule continuously increases. When the NO concentration is 70 μM, the fluorescence intensity increases by 14.7 times. Figure 3 This is the linear response graph of the probe to different NO concentrations. The fluorescence intensity shows a linear relationship with the NO concentration. The linear range is 3.0 - 70 μM, and the detection limit is 1.0 μM. This indicates that the probe can detect NO with high sensitivity.

[0040] Example 4:

[0041] Determination of the ultraviolet-visible absorption spectrum of the fluorescence probe interacting with NO

[0042] Figure 4 This is the ultraviolet-visible absorption spectrum diagram of the fluorescence probe after interacting with NO. The concentration of the fluorescence probe is 10 μM, and the added amount of NO is 70 μM. The instrument used for ultraviolet-visible absorption spectrum determination is the Agilent Cary60 ultraviolet-visible spectrophotometer. It can be seen from the figure that the probe does not have an obvious absorption band at 790 nm, and after adding NO, the absorption peak that appears near 790 nm gradually increases.

[0043] Example 5:

[0044] Selectivity of the fluorescence probe for NO determination

[0045] Figure 5 This is the selectivity graph of the fluorescence probe for NO determination. It is investigated that in a 10 μM fluorescence probe solution, NO (70 μM) and inorganic ions (K + , Ca 2+ , Na + , Mg 2+ , Ag + , Cu 2+ , Fe 3+ , NH4 + , Zn 2+ , CO3 2- , SO4 2- , Cl -,OH - ), reactive oxygen species (H2O2, ClO - ), reactive nitrogen species (NO2 - , ONOO - ) and biological thiols (Cys, Hcy, GSH). It can be seen from the figure that only NO can cause changes in the fluorescence spectrum, and other analytes have no obvious effect on the fluorescence spectrum of the probe. These results indicate that the fluorescent probe has good selectivity for NO.

[0046] Example 6:

[0047] Effect of solution pH on the fluorescence properties of the fluorescent probe for NO determination

[0048] The effect of pH on the fluorescence spectrum of the fluorescent probe for NO determination was investigated, and the results are as Figure 6 . The pH range we studied was 4.0 - 11.0, the concentration of the fluorescent probe was 10 μM, and the concentration of NO was 70 μM. It can be seen from the figure that as the pH increases, the fluorescence of the fluorescent probe is always in the off state, indicating that pH has little effect on the probe itself. However, after adding NO, as the pH increases, the fluorescence intensity increases significantly. When the pH reaches 8.0 and above, the fluorescence intensity gradually decreases. But within the physiological pH range of 7.0 - 8.0, the fluorescence intensity is basically stable. In summary, this probe has the potential for biological sample testing.

[0049] Example 7:

[0050] Determination of the response time of the fluorescent probe to NO

[0051] We studied the response time of the fluorescent probe to NO, and the results are as Figure 7 . It can be seen from the figure that the response time of this probe to NO is 150 s, which can meet the requirements for real-time monitoring in actual samples. It can also be seen from Figure 7 that after the fluorescence intensity reaches the maximum value, the fluorescence intensity does not change in the subsequent time, indicating that this fluorescent probe has good photostability.

[0052] Example 8:

[0053] Application of the fluorescent probe in living cells

[0054] First, we conducted a cytotoxicity test, as shown in Figure 8 . When 0 - 30 μM of the fluorescent probe was added, the cell viability was above 90%. This shows that the fluorescent probe has low toxicity and can be applied to detect NO in living cells. Then, the application of the fluorescent probe in living cells was studied, and macrophages were selected for confocal microscopy imaging, and the results are as Figure 9As shown, after the cells were stained with the probe Cy-Ac-NO for 30 min, the fluorescence was significantly enhanced. Subsequently, the cells were treated with lipopolysaccharide (LPS) for 12 h, and then stained with the probe Cy-Ac-NO for 30 min for imaging, and it was found that the fluorescence was significantly enhanced. After the cells were treated with lipopolysaccharide (LPS) and N-nitro-L-arginine (L-NNA) for 12 h, the probe Cy-Ac-NO was used for staining for 30 min for imaging, and weak fluorescence was produced. Figure 10 It is the relative fluorescence intensity map of the cells. These results indicate that the probe Cy-Ac-NO can sensitively detect NO in cells, thus providing a reliable means for monitoring the content of NO in biological samples.

Claims

1. A water-soluble nitric oxide fluorescent probe based on cyanine dye, namely Cy-Ac-NO, characterized in that, The structure is as follows:

2. The preparation method of a water-soluble nitric oxide fluorescent probe based on cyanine dye according to claim 1, wherein The reaction steps are as follows: Dissolve 1 equivalent of Cy-Ac-Cl in 15 - 25 mL of anhydrous acetonitrile, add it to a 50 mL round-bottom flask, slowly add 4 - 6 equivalents of triethylamine and 4 - 6 equivalents of 3-dimethylaminopropylamine, stir at 60 - 65 °C for 4 - 6 h under nitrogen protection; after the reaction is completed, remove the solvent from the crude product under reduced pressure, and purify it with an eluent of CH2Cl2 / CH3OH with a volume ratio of 25:1 - 20:1 to obtain the blue solid compound Cy-Ac-NO, which is the fluorescent probe described above. Among them, the structure of Cy-Ac-Cl is as follows:

3. Use of a water-soluble nitric oxide fluorescent probe based on cyanine dye according to claim 1, characterized in that, The fluorescent probe is applied to the detection of the content of nitric oxide in cells.

Images