A ratiometric nitric oxide-responsive near-infrared fluorescent molecular probe, its preparation method and application

Abstract

This invention discloses a ratiometric nitric oxide-responsive near-infrared fluorescent molecular probe, its preparation method, and its application. The near-infrared fluorescent molecular probe has a nitric oxide-responsive mechanism, with an electron-donating group consisting of an oxanthracene group linked to two amino groups. When the probe molecule interacts with the analyte, the diaminophenyl group of the hemicyanine core undergoes cyclization to form a triazole-containing ring, thereby causing intramolecular charge transfer and resulting in a ratiometric change in its corresponding ultraviolet absorption and fluorescence emission spectra. The near-infrared fluorescent molecular probe of this invention exhibits a selective response to nitric oxide, effectively reducing background signal interference and can be applied to the accurate detection of NO.

Description

Technical Field

[0001] This invention relates to a ratiometric near-infrared fluorescent molecular probe, particularly a ratiometric nitric oxide-responsive near-infrared fluorescent molecular probe, and also to the preparation method and application of the above-mentioned fluorescent molecular probe. Background Technology

[0002] Nitric oxide (NO) is widely present in the tissues of many mammals. As a biological signaling molecule, it is an important regulator of vital activities, participating not only in the regulation of the cardiovascular system but also in the transmission of biological signals in the nervous and immune systems, exhibiting antibacterial, antitumor, vasodilatory, and wound-healing effects. Particularly in the immune system, NO is considered a key factor in protecting the body from external pathogens. However, NO is a gaseous free radical, and when its levels are too high, it can combine with other free radicals and important substances such as proteins in the body, thereby causing damage. For example, in physiological disorders, activated macrophages produce high levels of NO, which upregulates osteoclasts, destroys cartilage, and recruits other immune cells, causing inflammation and joint damage. Therefore, real-time monitoring of NO concentration in the body is crucial.

[0003] Molecular fluorescent probes have attracted much attention due to their high sensitivity and selectivity, enabling accurate identification of target analytes and non-invasive real-time in vivo observation. Currently reported NO probes mainly consist of o-phenylenediamine and aromatic secondary amines, which can specifically react with NO to exhibit an "on" fluorescent signal. These probes are typically based on the photoinduced electron transfer (PET) effect, i.e., enhanced fluorescence in the presence of NO, and can only be used for qualitative detection. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a ratiometric nitric oxide-responsive near-infrared fluorescent molecular probe based on the o-phenylenediamine structure and exhibiting the ICT effect, and also to provide the preparation method and application of the above-mentioned fluorescent molecular probe.

[0005] Technical solution: The present invention provides a ratiometric nitric oxide-responsive near-infrared fluorescent molecular probe, with the following structural formula:

[0006]

[0007] Among them, R1, R2, R3, and R4 are H or NH2.

[0008] The structural formula of the molecular probe is as follows:

[0009]

[0010] Among them, R1 and R4 are H, and R2 and R3 are NH2; or R1 and R2 are NH2, and R3 and R4 are H; or R1 and R2 are H, and R3 and R4 are NH2.

[0011] The preparation method of the above-mentioned molecular probe includes the following steps:

[0012] (1) Mix compound 1 and compound 2, add N,N-dimethylformamide under inert gas protection, stir until uniform, add triethylamine, stir the mixture and filter and dry to obtain intermediate compound, wherein compound 2 is one of 4-amino-3-nitrophenol, 2-amino-3-nitrophenol or 3-amino-4-nitrophenol;

[0013] (2) Dissolve the intermediate compound in anhydrous methanol, add hydrochloric acid solution of stannous chloride dihydrate dropwise under inert gas protection, heat to reflux, cool after the reaction is completed, distill under reduced pressure, and obtain the near-infrared fluorescent molecular probe by silica gel column chromatography.

[0014]

[0015] In step (1), the molar ratio of compound 1, compound 2 and triethylamine is 1:3-5:3-5.

[0016] In step (1), the ratio of compound 1 to N,N-dimethylformamide is 1 mmol: 10-15 mL.

[0017] In step (2), the heating reflux reaction is carried out at a temperature of 70-90℃ for 7-9 hours.

[0018] In step (2), the silica gel column has a mesh size of 200-300; the eluent is a mixture of dichloromethane and methanol with a volume ratio of 80:1 to 100:1.

[0019] In step (2), the solution turns green at the end of the reaction.

[0020] The application of the above-mentioned near-infrared fluorescent molecular probes in the detection of NO.

[0021] The near-infrared fluorescent molecular probe is used in reagents or kits for the quantitative detection of NO in solution, cells, or living organisms.

[0022] Invention Principle: The fluorescent molecule of this invention has a hemicyanine near-infrared fluorophore as its core, with a diaminophenyl group as the NO recognition group. It can absorb near-infrared light and emit fluorescence with a longer wavelength. Compared with the single-channel fluorescence imaging mode of "on" probes, the ratiometric fluorescent probe based on the intramolecular charge transfer (ICT) effect can perform quantitative detection by ratio, which can effectively reduce background signal interference. It is preferred for high-resolution, self-calibrated NO imaging, realizing accurate spatiotemporal detection of NO.

[0023] Because the hemicyanine nucleus and the recognition group are covalently bonded to form a conjugated system, and the electron-donating groups in this structure are the oxanthracene groups connected to the two amino groups, when the probe molecule interacts with the analyte, the diaminophenyl group of the hemicyanine nucleus undergoes cyclization to form a triazole-containing ring, thereby causing intramolecular charge transfer. This results in ratiometric changes in the corresponding ultraviolet absorption and fluorescence emission spectra, thus achieving accurate detection of NO. Furthermore, the near-infrared properties of this probe can improve the detection depth and resolution.

[0024] Meanwhile, this probe is a ratiometric fluorescent probe that can self-calibrate its two emission peaks, thereby eliminating the influence of environmental factors on experimental results (including human interference caused by changes in the probe environment, probe concentration, and excitation intensity), and increasing the effective detection range. Significant results from cell and in vivo imaging experiments demonstrate the potential of this probe for clinical applications.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following advantages: (1) The present invention uses diaminophenyl as the responsive group for NO detection, which has excellent selectivity, fast reaction rate, low toxicity, and good ratiometric imaging effect at the cellular level; (2) The near-infrared fluorescent probe of the present invention absorbs and emits fluorescence in the near-infrared region, which has deeper tissue penetration, and the ratiometric imaging characteristics of the probe improve its sensitivity and accuracy and reduce background interference. Attached Figure Description

[0026] Figure 1 This is a graph showing the ultraviolet absorption and fluorescence changes of the near-infrared fluorescent probe of the present invention in response to nitric oxide;

[0027] Figure 2 This is a graph showing the MTT experimental data of the near-infrared fluorescent probe of this invention, with the horizontal axis in μM.

[0028] Figure 3 This is a confocal microscopy cell imaging image of the near-infrared fluorescent probe of the present invention. Detailed Implementation

[0029] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments. All materials are commercially available.

[0030] Example 1

[0031] Preparation of ratiometric nitric oxide-responsive near-infrared fluorescent molecular probe of formula 1:

[0032] (1) Take 1 mol of compound 1 and 5 mol of 4-amino-3-nitrophenol and add them to a round-bottom flask. Under nitrogen protection, slowly add N,N-dimethylformamide. The ratio of compound 1 to N,N-dimethylformamide is 1 mmol: 12 mL. Stir well and slowly add 5 mol of triethylamine. After stirring for 5 h, filter and dry the mixture to obtain the intermediate compound.

[0033] (2) The intermediate compound was dissolved in anhydrous methanol, and 5 mol of hydrochloric acid solution of SnCl2·2H2O was slowly added dropwise to the reaction solution under nitrogen protection. The mixture was heated under reflux for 8 h, cooled, distilled under reduced pressure, and purified by silica gel column chromatography to obtain the near-infrared fluorescent molecular probe of formula 1. 1 H NMR(300MHz,Methanol-d4)δ8.41(d,1H),8.12(d,1H),7.91(dd,2H),7.54(m,1H),7.49-7.33(m,3H),6.77 (s,1H),6.67(s,1H),5.97(d,1H),4.14(q,2H),2.66(dt,4H),1.94(s,6H),1.90-1.78(m,2H),1.37(t,3H).

[0034] Example 2

[0035] Preparation of ratiometric nitric oxide-responsive near-infrared fluorescent molecular probe formula 2:

[0036] (1) Take 1 mol of compound 1 and 5 mol of 2-amino-3-nitrophenol and add them to a round-bottom flask. Under nitrogen protection, slowly add N,N-dimethylformamide. The ratio of compound 1 to N,N-dimethylformamide is 1 mmol: 12 mL. Stir well and slowly add 5 mol of triethylamine. After stirring for 5 h, filter and dry the mixture to obtain the intermediate compound.

[0037] (2) The intermediate compound was dissolved in anhydrous methanol, and 5 mol of hydrochloric acid solution of SnCl2·2H2O was slowly added dropwise to the reaction solution under nitrogen protection. The mixture was heated under reflux for 8 h, cooled, distilled under reduced pressure, and purified by silica gel column chromatography to obtain the near-infrared fluorescent molecular probe formula 2. 1H NMR(300MHz,Methanol-d4)δ8.45(d,1H),8.15(d,1H),7.85(dd,2H),7.63(m,1H),7.52-7.35(m,3H),6.52 (d,1H),6.33(d,1H),5.87(d,1H),4.14(q,2H),2.68(dt,4H),1.95(s,6H),1.91-1.75(m,2H),1.37(t,3H).

[0038] Example 3

[0039] Preparation of ratiometric nitric oxide-responsive near-infrared fluorescent molecular probe formula 3:

[0040] (1) Take 1 mol of compound 1 and 5 mol of 3-amino-4-nitrophenol and add them to a round-bottom flask. Under nitrogen protection, slowly add N,N-dimethylformamide. The ratio of compound 1 to N,N-dimethylformamide is 1 mmol: 12 mL. Stir well and slowly add 5 mol of triethylamine. After stirring for 5 h, filter and dry the mixture to obtain the intermediate compound.

[0041] (2) The intermediate compound was dissolved in anhydrous methanol, and 5 mol of hydrochloric acid solution of SnCl2·2H2O was slowly added dropwise to the reaction solution under nitrogen protection. The mixture was heated under reflux for about 8 hours, cooled, distilled under reduced pressure, and purified by silica gel column chromatography to obtain the near-infrared fluorescent molecular probe of formula 3. 1 H NMR(300MHz,Methanol-d4)δ8.52(d,1H),8.23(d,1H),7.78(dd,2H),7.66(m,1H),7.67-7.49(m,3H),7.32 (d,1H),7.12(d,1H),5.97(d,1H),4.16(q,2H),2.68(dt,4H),1.95(s,6H),1.92-1.83(m,2H),1.36(t,3H).

[0042] Example 4

[0043] Experiments on the in vitro response of near-infrared fluorescent probes to nitric oxide in ultraviolet absorption and fluorescence emission:

[0044] Weigh 1.21 mg of the probe prepared in Example 1 and dissolve it in 1 mL of DMSO to prepare a 10 mM probe stock solution.

[0045] The probe NFL-NH2 (10 μM) and the NO donor (DEANONOate, 0–60 μM) were stored at 37 °C for 2 minutes. Absorption and fluorescence spectra were obtained using a UV-Vis spectrophotometer and a fluorescence spectrophotometer, respectively (excitation wavelength 650 nm). The data were then processed using Origin software. Figure 1 In the UV spectrum of the reaction between NFL-NH2 solution and nitric oxide, the absorption peaks exhibit a ratiometric change. Specifically, after the addition of nitric oxide donor, the absorption peak at 740 nm gradually decreases, while the absorption peak at 625 nm gradually increases. Correspondingly, with the addition of NO, the fluorescence value at 780 nm gradually decreases, while the fluorescence value at 705 nm gradually increases, indicating that the probe responds well to nitric oxide in solution.

[0046] Example 5

[0047] MTT biocompatibility assay of near-infrared fluorescent probes with RAW 264.7 cells:

[0048] RAW 264.7 cells were seeded into the remaining wells of a 96-well plate by adding PBS solution around the outer edge and incubating for 12 hours. 3.0 mg of the probe was dissolved in 5 mL of biological DMSO solution to prepare a 5 mM biological probe stock solution. A 5 mg / mL LMT solution was prepared. Seven 5 mL centrifuge tubes were prepared, and the probe concentrations were serially diluted to 0, 5, 10, 15, 20, 25, and 30 μM, respectively. 190 μL of each solution was added to the wells in a horizontal row, with equal concentrations, and incubated for 24 hours. The culture medium was removed, and the cells were washed with PBS. 10 μL of LMT solution was added to each well, and incubation continued for 4 hours. The culture medium was discarded, and 100 μL of DMSO solution was added to each well. After complete dissolution, UV absorption was performed (detection wavelength 490 nm).

[0049] Data processing using Origin Figure 2 It can be seen that when the probe concentration reaches 30 μM, the cell survival rate is higher than 80% after 24 hours of incubation, indicating that the probe has good biosafety and low cytotoxicity.

[0050] Example 6

[0051] Confocal microscopy cell experiments using near-infrared fluorescent probes and nitric oxide responses:

[0052] RAW 264.7 cells at appropriate concentrations were seeded in three confocal microscopy dishes and cultured overnight at 37°C under a 5% CO2 atmosphere. Two dishes were selected; one dish was treated with the inflammation-inducing agent LPS, and the other dish was treated with both LPS and the nitric oxide synthase inhibitor AG. Finally, the cells were incubated with the probe for 5 minutes, washed three times with PBS, and then subjected to laser confocal imaging using the green fluorescence channel (λ). em =680-720nm) and red fluorescence channel (λ) em Confocal fluorescence imaging was performed at 750-790 nm.

[0053] Images taken with a confocal microscope, such as Figure 3 Compared to the control group, LPS stimulation of cells resulted in stronger fluorescence intensity and clearer imaging in the green fluorescence channel. The green fluorescence signal in the LPS+AG group was weakened compared to the LPS group. All three cell images were clear, demonstrating excellent ratiometric fluorescence change imaging of the probe within the cells.

Claims

1. A ratiometric nitric oxide-responsive near-infrared fluorescent molecular probe, characterized in that, The structural formula of the molecular probe is as follows:

2. A method for preparing the molecular probe according to claim 1, characterized in that, Includes the following steps: (1) Mix compound 1 and compound 2, add N,N-dimethylformamide under inert gas protection, stir until uniform, add triethylamine, stir the mixture and filter and dry to obtain intermediate compound, wherein compound 2 is one of 4-amino-3-nitrophenol, 2-amino-3-nitrophenol or 3-amino-4-nitrophenol; (2) Dissolve the intermediate compound in anhydrous methanol, add hydrochloric acid solution of stannous chloride dihydrate dropwise under inert gas protection, heat to reflux, cool after the reaction is completed, distill under reduced pressure, and obtain the near-infrared fluorescent molecular probe by silica gel column chromatography.

3. The manufacturing method according to claim 2, characterized in that, In step (1), the molar ratio of compound 1, compound 2 and triethylamine is 1:3 to 5:3 to 5.

4. The manufacturing method according to claim 2, characterized in that, In step (1), the ratio of compound 1 to N,N-dimethylformamide is 1 mmol: 10-15 mL.

5. The manufacturing method according to claim 2, characterized in that, In step (2), the heating reflux reaction is carried out at a temperature of 70-90℃ for 7-9 hours.

6. The manufacturing method according to claim 2, characterized in that, In step (2), the silica gel column is 200-300 mesh; the eluent is a mixture of dichloromethane and methanol with a volume ratio of 80:1 to 100:

1.

7. The manufacturing method according to claim 2, characterized in that, In step (2), the solution turns green at the end of the reaction.

8. The application of the near-infrared fluorescent molecular probe of claim 1 in the preparation of a solution or cell-based quantitative detection reagent for NO.

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