Near-infrared fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a near-infrared fluorescent probe as well as a preparation method and application thereof. The near-infrared fluorescent probe has a structure as shown in a compound 1. The fluorescent probe obtained by the preparation method has stronger ultraviolet in a near-infrared region; cysteine can be rapidly and efficiently detected in a mixed solution formed by N, N-dimethylformamide and water, and a new absorption peak is generated at 750nm. The fluorescent probe obtained by the preparation method has stronger fluorescence in a near-infrared region; the cysteine can be rapidly and efficiently detected in a mixed solution formed by N, N-dimethylformamide and water, and the fluorescence at 702nm can be gradually quenched along with the increase of the concentration of the cysteine.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes and relates to a near-infrared fluorescent probe and a preparation method and application thereof. Background Art

[0002] Biothiols, such as glutathione (GSH) and cysteine ​​(Cys), are important biomarkers that are closely related to many diseases and play a vital role in many physiological processes. Therefore, the detection of biothiols is very important for the early diagnosis of diseases and the assessment of disease progression. Cysteine ​​is an essential amino acid in the human body. It participates in the reduction process of cells and phospholipid metabolism in the liver, and has the function of protecting liver cells from damage. It is also an important component of glutathione. It can protect the defense system from oxidation, affect the formation, folding, assembly and stability of disulfide bonds, and is of great significance to the antioxidant and free radical elimination functions of organisms. In addition, due to its binding effect in the body, it has been proven to be an effective detoxifier. Therefore, seeking an efficient and rapid method for detecting cysteine ​​is of great significance for the diagnosis of early diseases in organisms.

[0003] At present, there are several methods for the detection of cysteine: (1) titration method, (2) ultraviolet absorption method, (3) molecular fluorescence analysis method, (4) emission spectroscopy analysis method, and (5) mass spectrometry method. The selectivity of titration method and emission spectroscopy method is not high, and the sensitivity is low, while ultraviolet absorption method and mass spectrometry method have high requirements for instruments and relatively high analysis costs. Compared with several methods, the fluorescent probe method in molecular fluorescence analysis has obvious advantages, including high sensitivity, good selectivity, and low cost. PPAB (pyrrolopyrrole aza-BODIPY) has the characteristics of strong absorption, strong emission, high extinction coefficient, high fluorescence quantum yield, and stable photothermal physical properties in the near-infrared spectral region, and has unique advantages in the field of biological imaging.

[0004] We modified PPAB to connect aldehyde groups at both ends to detect cysteine, and modified its raw materials with alkoxy chains to increase the overall solubility of the compound and improve its yield. Through selectivity testing, we found that it has good selectivity for cysteine ​​and due to its excellent optical properties in the near-infrared, we successfully applied its sensing performance to cell fluorescence imaging. Summary of the invention

[0005] Technical problem to be solved: The present invention provides a near-infrared fluorescent probe and a preparation method and application thereof, which can be used to detect cysteine.

[0006] Technical solution: A near-infrared fluorescent probe having the structure shown in compound 1:

[0007]

[0008] The synthesis method of the near-infrared fluorescent probe comprises the following steps: step 1, adding 1 equivalent of DPP-Br and 2 to 6 equivalents of 2-amino-3-((2-hexyldecyl)oxy)pyridine to dry toluene, refluxing at 110°C under N2 protection, adding 5 to 10 equivalents of TiCl4 to the mixed solution after it is fully dissolved, continuing to reflux and stir for 10 to 15 minutes, and then adding 20 to 25 equivalents of NEt3; continuing to reflux and stir for 2 to 3 hours, and adding 22 to 37 equivalents of BF3·OEt2 after the formation of the intermediate product imine is monitored by thin layer chromatography silica gel plate, and then continuing to reflux for 12 to 1 5h; cooled to room temperature, poured the reaction mixture into water, and extracted with CH2Cl2; added anhydrous sodium sulfate to the organic layer, dried, filtered, and rotary evaporated to remove CH2Cl2, and the crude product was separated by column chromatography to obtain compound 2; step 2, 1 equivalent of compound 2, 0.2-0.5 equivalent of Pd(dppf)Cl2·CH2Cl2, 15-20 equivalents of KOAc and 3-5 equivalents of 4-formylphenylboronic acid were added to dry DMF, refluxed in anhydrous and oxygen-free environment under N2 protection, cooled to room temperature, evaporated under reduced pressure, removed the solvent, and then separated by column chromatography to obtain compound 1.

[0009] Preferably, the molar ratio of DPP-Br to 2-amino-3-((2-hexyldecyl)oxy)pyridine is 1:5.

[0010] Preferably, the amount of TiCl4 added is 6 equivalents.

[0011] Preferably, the amount of NEt3 added is 20 equivalents.

[0012] Preferably, the molar ratio of the compound 2, Pd(dppf)Cl2·CH2Cl2, KOAc and 4-formylphenylboronic acid added is 1:0.26:16:1.2.

[0013] Preferably, the reflux temperature in the above step 2 is 80° C., and the reflux time is 3 to 4 hours.

[0014] The compound 1 is used in the preparation of a kit for detecting intracellular cysteine.

[0015] A kit for detecting cysteine ​​contains the above-mentioned near-infrared fluorescent probe and N-ethylmaleimide.

[0016] The reaction formula is as follows:

[0017]

[0018] Beneficial effects: A fluorescent probe for detecting cysteine ​​based on the present invention connects phenylboronic acid with an aldehyde group to a luminescent group through a Suzuki reaction. The preparation method uses 4-formylphenylboronic acid to undergo a coupling reaction with a luminescent group PPAB-Br containing bromine atoms at both ends. The raw materials are easily available, the method is simple, and the obtained product is a green solid powder, which is easy to store and has good stability. The fluorescent probe obtained by the preparation method has strong ultraviolet in the near-infrared region; in a mixed solution formed by N, N-dimethylformamide and water, cysteine ​​can be detected quickly and efficiently and a new absorption peak is generated at 750nm. The fluorescent probe obtained by the preparation method has strong fluorescence in the near-infrared region; in a mixed solution formed by N, N-dimethylformamide and water, cysteine ​​can be detected quickly and efficiently and its fluorescence at 702nm will be gradually quenched as the concentration of cysteine ​​increases. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the mass HRMS spectrum of the fluorescent probe compound 1 for detecting cysteine ​​prepared in Example 1.

[0020] Figure 2 This is the UV image of the fluorescent probe compound 1 for detecting cysteine ​​prepared in Example 1 in dichloromethane.

[0021] Figure 3 This is a fluorescence image of a fluorescent probe compound 1 for detecting cysteine ​​prepared in Example 1 in dichloromethane.

[0022] Figure 4 The absorption spectrum change of probe compound 1 (10 μM) after reacting with cysteine ​​(10 μM) for 2 min, the abscissa is wavelength (nm), and the ordinate is absorbance (Abs).

[0023] Figure 5 The fluorescence spectrum change of the probe compound 1 (10 μM) after reacting with cysteine ​​(10 μM) for 2 minutes, the excitation wavelength is 630 nm, the abscissa is the wavelength (nm), and the ordinate is the fluorescence intensity (FL).

[0024] Figure 6 The fluorescence change of compound 1 (10 μM) at 675 nm in the presence of cysteine ​​and other competing species (100 μM), including the reaction species, 1, glutathione, 2, glycine, 3, serine, 4, glutamine, 5, glutamate, 6, methionine, and the selectivity after response.

[0025] Figure 7The UV changes of compound 1 (10 μM) at 702 nm in the presence of cysteine ​​and other competing species (100 μM) include reactive species, 1, glutathione, 2, glycine, 3, serine, 4, glutamine, 5, glutamate, 6, methionine, and after response, the selectivity at an excitation wavelength of 630 nm.

[0026] Figure 8 Fluorescence imaging of HeLa cells incubated with probe compound 1, cysteine ​​and NEM (thiol blocking agent) under the red light channel. First row: Fluorescence imaging of HeLa cells in bright field and red light channel respectively when only probe compound 1 (1 μM) was incubated; second row: Fluorescence imaging of HeLa cells in bright field and red light channel respectively when probe compound 1 (1 μM) and cysteine ​​(1 μM) were co-incubated; third row: Fluorescence imaging of HeLa cells in bright field and red light channel respectively when probe compound 1 (1 μM) and NEM (1 μM) were co-incubated. DETAILED DESCRIPTION

[0027] The following examples are given to illustrate the present invention in more detail. It is necessary to point out that the following examples cannot be interpreted as limiting the scope of protection of the invention. Some non-essential improvements and adjustments made to the present invention by technical personnel skilled in the art based on the above invention content should still fall within the scope of protection of the present invention.

[0028] The experimental methods used in the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the experiments can be obtained from commercial sources unless otherwise specified. All the following reagents used in the examples are commercially available analytically pure or chemically pure.

[0029] Example 1

[0030] Based on compound 1 with aldehyde groups at both ends, it was prepared by the following method:

[0031] (1) Preparation of Compound 2

[0032] DPP-Br (440 mg, 5 mmol) and 2-amino-3-((2-hexyldecyl)oxy)pyridine (1.5 g, 4.5 mmol) were added to dry toluene (30 mL), refluxed at 110 ° C under N2 protection, and TiCl4 (0.6 mL, 5.4 mmol) was added to the mixed solution after it was fully dissolved. After continuing to reflux and stir for 10 to 15 minutes, NEt3 (2.0 mL, 14 mmol) was added. Reflux and stir for about 2 to 3 hours. After monitoring the formation of the intermediate product imine by thin layer chromatography silica gel plate, boron trifluoride etherate (1.8 mL, 15 mmol) was added, and then refluxed for 12 to 15 hours to obtain a dark blue mixed solution. Cool to room temperature, pour the reaction mixture into water, and extract with CH2Cl2. The organic layer was dried by adding anhydrous sodium sulfate, filtered, and CH2Cl2 was removed by rotary evaporation. The crude product was separated by column chromatography to obtain compound 2 (433 mg, yield 73%); 1 H NMR (500MHz, Chloroform-d): δ [ppm] = 8.34 (d, J = 9.5Hz, 4H), 7.87 (d, J = 6.0Hz, 2H), 7.65 (d, J = 8.5Hz, 4H), 7.25 (d, J = 9.5Hz, 2H), 7.05 (dd, J = 6.0Hz, J = 8.0Hz 2H).

[0033] The structural formula of the obtained compound 2 is:

[0034]

[0035] (2) Preparation of Compound 1

[0036] Compound 2 (25 mg, 0.021 mmol), Pd(dppf)Cl2 CH2Cl2 (4.6 mg, 0.26 eq.), KOAc (34 mg, 16 eq.) and 4-formylphenylboronic acid (40 mg, 1.2 eq.) were added to dry DMF (1.6 mL). Refluxed for 3-4 h at 80 ° C under N2 protection in anhydrous and oxygen-free conditions. Cooled to room temperature, evaporated under pressure, the solvent was removed, and then separated by column chromatography to obtain compound 1 (5 mg, yield 10.5%); 1 H NMR (500MHz, Chloroform-d): δ = 10.08 (s, 2H), 8.56 (d, 4H), 7.87 (d, 2H), 7.71 (d, 4H), 7.58 (d, 4H), 7.19 (d, 2H), 6.99 (dd, 2H), 6.91 (d, 4H).

[0037] The structural formula of the obtained compound 1 is:

[0038]

[0039] The mass spectrometry HRMS spectrum of probe compound 1 is shown in Figure 1 .

[0040] The absorption and fluorescence spectra of probe compound 1 are shown in Figure 2 and Figure 3 .

[0041] Sensing performance of probe compound 1 towards cysteine

[0042] The prepared fluorescent probe compound 1 was dissolved in N,N-dimethylformamide solution, and the sensing performance of compound 1 to cysteine ​​was tested. In the presence of cysteine ​​(10 μM), it was observed that the UV-Vis absorption of the probe at 675 nm decreased after 2 minutes, while the UV-Vis absorption at 750 nm increased. Figure 4 This change in UV-vis absorption is accompanied by a gradual quenching of the near-infrared fluorescence emission at 702 nm as shown in Figure 5 shown.

[0043] Example 2

[0044] Selectivity of probe compound 1

[0045] To test the selectivity of compound 1 for cysteine, the spectral changes of the probe in the presence of cysteine ​​or other potential biothiols (including glycine, glutamate, serine, methionine, glutamine, glutathione, etc.) were detected.

[0046] like Figure 6 As shown in the figure, the near-infrared fluorescence quenching of compound 1 occurs within 2 minutes only in the presence of cysteine, and the fluorescence attenuation caused by other analytes is very small. The new peak generated by compound 1 at 750nm is only produced in the presence of cysteine, and other analytes have basically no such effect. Figure 7 shown.

[0047] Example 3

[0048] Application of probe compound 1 in detecting cysteine ​​in cells

[0049] HeLa cells were incubated with probe compound 1, probe compound 1 and cysteine, and compound 1 and NEM, respectively. The results are shown in Figure 8As shown, through observation, when the cells were incubated with probe compound 1 only, the cells emitted weak fluorescence due to the presence of certain cysteine ​​in the cells; when the cells were incubated with compound 1 and cysteine, the fluorescence in the cells was quenched; however, when the cells were incubated with compound 1 and NEM, since NEM is an effective thiol blocking agent, the fluorescence in HeLa cells was significantly enhanced compared with the first two groups, indicating that the detection of cysteine ​​by probe compound 1 can be applied to cells.

[0050] To sum up, the above are only preferred specific implementation modes of the present invention, and the protection scope of the present invention is not limited thereto. Any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by any technician familiar with the technical field within the technical scope disclosed in the present invention all fall within the protection scope of the present invention.

Claims

1. A near-infrared fluorescent probe, characterized in that: It has the structure shown in compound 1:

2. The method for synthesizing the near-infrared fluorescent probe according to claim 1, characterized in that: The method comprises the following steps: step 1, adding 1 equivalent of DPP-Br and 2 to 6 equivalents of 2-amino-3-((2-hexyldecyl)oxy)pyridine into dry toluene, refluxing at 110°C under N2 protection, adding 5 to 10 equivalents of TiCl4 into the mixed solution after being fully dissolved, continuing to reflux and stir for 10 to 15 minutes, and then adding 20 to 25 equivalents of NEt3; continuing to reflux and stir for 2 to 3 hours, adding 22 to 37 equivalents of BF3·OEt2 after monitoring the formation of the intermediate product imine through a thin layer chromatography silica gel plate, and then continuing to reflux for 12 to 15 hours; cooling to room temperature , pour the reaction mixture into water and extract with CH2Cl2; add anhydrous sodium sulfate to the organic layer, dry it, filter, and remove CH2Cl2 by rotary evaporation. The crude product is separated by column chromatography to obtain compound 2; step 2, add 1 equivalent of compound 2, 0.2-0.5 equivalent of Pd(dppf)Cl2·CH2Cl2, 15-20 equivalents of KOAc and 3-5 equivalents of 4-formylphenylboronic acid to dry DMF, reflux in anhydrous and oxygen-free environment under N2 protection, cool to room temperature, evaporate under reduced pressure, remove the solvent, and then separate by column chromatography to obtain compound 1.

3. The method for synthesizing the near-infrared fluorescent probe according to claim 2, characterized in that: The molar ratio of the DPP-Br to 2-amino-3-((2-hexyldecyl)oxy)pyridine is 1:

5.

4. The method for synthesizing the near-infrared fluorescent probe according to claim 2, characterized in that: The added amount of TiCl4 is 6 equivalents.

5. The method for synthesizing the near-infrared fluorescent probe according to claim 2, characterized in that: The amount of NEt3 added is 20 equivalents.

6. The method for synthesizing the near-infrared fluorescent probe according to claim 2, characterized in that: The compound 2, The molar ratio of Pd(dppf)Cl2·CH2Cl2, KOAc and 4-formylphenylboronic acid added was 1:0.26:16:1.

2.

7. The method for synthesizing the near-infrared fluorescent probe according to claim 2, characterized in that: The reflux temperature in step 2 is 80° C., and the reflux is performed for 3 to 4 hours.

8. Use of the compound 1 according to claim 1 in the preparation of a kit for detecting intracellular cysteine.

9. A kit for detecting cysteine, characterized in that: Contains the near-infrared fluorescent probe according to claim 1 and N-ethylmaleimide.

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