A near-infrared fluorescent probe and a preparation method and application thereof

Abstract

The application discloses a near-infrared fluorescent probe, a preparation method and application thereof, and has the structure shown in compound 1. The fluorescent probe prepared by the preparation method has strong ultraviolet in the near-infrared region; and can rapidly and efficiently detect cysteine in a mixed solution formed by N,N dimethylformamide and water and generate a new absorption peak at 750 nm. The fluorescent probe prepared by the preparation method has strong fluorescence in the near-infrared region; and can rapidly and efficiently detect cysteine in a mixed solution formed by N,N dimethylformamide and water and generate a new absorption peak at 750 nm.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, and relates to a near-infrared fluorescent probe, its preparation method and application. Background Technology

[0002] Biothiols, such as glutathione (GSH) and cysteine ​​(Cys), are important biomarkers closely related to many diseases and play a crucial role in many physiological processes. Therefore, the detection of biothiols is very important for the early diagnosis and assessment of disease progression. Cysteine ​​is an essential amino acid in the human body, participating in cellular reduction processes and phospholipid metabolism in the liver, and playing a protective role against hepatocyte damage. It is also an important component of glutathione, which protects the defense system from oxidation, affects the formation, folding, assembly, and stability of disulfide bonds, and is of great significance for the body's antioxidant and free radical scavenging functions. In addition, due to its binding effect in the body, it has been proven to be an effective detoxifying agent. Therefore, seeking an efficient and rapid method for detecting cysteine ​​is of great significance for the early diagnosis of diseases.

[0003] Currently, there are several methods for detecting cysteine: (1) titration, (2) ultraviolet absorption, (3) molecular fluorescence analysis, (4) emission spectroscopy, and (5) mass spectrometry. Titration and emission spectroscopy have low selectivity and low sensitivity, while ultraviolet absorption and mass spectrometry require high-performance instruments and are relatively expensive. Among these methods, the fluorescent probe method in molecular fluorescence analysis has significant advantages, including high sensitivity, good selectivity, and low cost. PPAB (pyrrolopyrrolaza-BODIPY) has strong absorption, strong emission, high extinction coefficient, high fluorescence quantum yield, and stable photothermal properties in the near-infrared spectral region, giving it unique advantages in the field of bioimaging.

[0004] We modified PPAB by attaching aldehyde groups to both ends to detect cysteine. Adding alkoxy groups to the starting material increased the overall solubility of the compound and improved its yield. Through selectivity testing, we found it to have good selectivity for cysteine, and due to its excellent near-infrared optical properties, we successfully applied its sensing properties to cell fluorescence imaging. Summary of the Invention

[0005] Technical problem solved: This invention provides a near-infrared fluorescent probe, its preparation method and application, 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 above near-infrared fluorescent probe includes the following steps: Step 1, 1 equivalent of DPP-Br and 2-6 equivalents of 2-amino-3-((2-hexyldecyl)oxy)pyridine are added to dry toluene, and refluxed at 110°C under N2 protection. After complete dissolution, 5-10 equivalents of TiCl4 are added to the mixed solution, and reflux and stirring are continued for 10-15 min. Then, 20-25 equivalents of NEt3 are added; reflux and stirring are continued for 2-3 h. After the formation of the intermediate imine is monitored by thin-layer chromatography on silica gel plate, 22-37 equivalents of BF3·OEt2 are added, followed by reflux for 12-1 h. 5 h; Cool to room temperature, pour the reaction mixture into water, and extract with CH2Cl2; Dry the organic layer with anhydrous sodium sulfate, filter, remove CH2Cl2 by rotary evaporation, and separate the crude product by column chromatography to obtain compound 2; Step 2, add 1 equivalent of compound 2, 0.2-0.5 equivalents of Pd(dppf)Cl2·CH2Cl2, 15-20 equivalents of KOAc and 3-5 equivalents of 4-formylphenylboronic acid to dry DMF, reflux under anhydrous and oxygen-free N2 protection, cool to room temperature, evaporate under reduced pressure to remove solvent, and then separate 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 compound 2, Pd(dppf)Cl2·CH2Cl2, KOAc and 4-formylphenylboronic acid is 1:0.26:16:1.2.

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

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

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

[0016] The reaction formula is shown below:

[0017]

[0018] Beneficial Effects: This invention provides a fluorescent probe for detecting cysteine, which connects phenylboronic acid with an aldehyde group to a luminescent group via the Suzuki reaction. The preparation method uses 4-formylphenylboronic acid in a coupling reaction with a luminescent group PPAB-Br containing bromine atoms at both ends. The raw materials are readily available, the method is simple, and the resulting product is a green solid powder that is easy to store and has good stability. The fluorescent probe obtained by this method exhibits strong ultraviolet light in the near-infrared region; it can rapidly and efficiently detect cysteine ​​in a mixed solution of N,N-dimethylformamide and water, generating a new absorption peak at 750 nm. Furthermore, the fluorescence at 702 nm is gradually quenched as the concentration of cysteine ​​increases. Attached Figure Description

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

[0020] Figure 2 This is a UV image of a 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 changes of probe compound 1 (10 μM) after reacting with cysteine ​​(10 μM) for 2 min are shown on the x-axis, which represents wavelength (nm) and the y-axis represents absorbance (Abs).

[0023] Figure 5 The fluorescence spectrum changes of probe compound 1 (10 μM) after reacting with cysteine ​​(10 μM) for 2 min are shown. The excitation wavelength is 630 nm. The x-axis represents wavelength (nm) and the y-axis represents 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 responding species, 1, glutathione, 2, glycine, 3, serine, 4, glutamine, 5, glutamic acid, 6, methionine, was determined, along with the selectivity after the response.

[0025] Figure 7To determine the UV change of compound 1 (10 μM) at 702 nm in the presence of cysteine ​​and other competing species (100 μM), including the reactant species 1, glutathione, 2, glycine, 3, serine, 4, glutamine, 5, glutamic acid, and 6, methionine, the selectivity at an excitation wavelength of 630 nm was determined.

[0026] Figure 8 Fluorescence imaging of HeLa cells incubated with probe compound 1, cysteine, and NEM (thiol blocking agent) under red light. First row: Fluorescence imaging of HeLa cells incubated with probe compound 1 (1 μM) alone under bright field and red light. Second row: Fluorescence imaging of HeLa cells incubated with both probe compound 1 (1 μM) and cysteine ​​(1 μM) under bright field and red light. Third row: Fluorescence imaging of HeLa cells incubated with both probe compound 1 (1 μM) and NEM (1 μM) under bright field and red light. Detailed Implementation

[0027] The following examples illustrate the invention in more detail. It should be noted that the following embodiments should not be construed as limiting the scope of protection of the invention. Any non-essential improvements and adjustments made to the invention by those skilled in the art based on the above-described invention should still fall within the scope of protection of the invention.

[0028] Unless otherwise specified, the experimental methods used in this invention are conventional methods. Unless otherwise specified, all materials and reagents used in the experiments are commercially available. All reagents used in the examples are commercially available analytical grade or chemically pure.

[0029] Example 1

[0030] Compound 1, which has aldehyde groups at both ends, 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) and refluxed at 110 °C under N2 protection. After complete dissolution, TiCl4 (0.6 mL, 5.4 mmol) was added to the mixture, and the mixture was refluxed and stirred for 10–15 min. Then, NEt3 (2.0 mL, 14 mmol) was added. The mixture was refluxed and stirred for about 2–3 h. After the formation of the intermediate imine was detected by silica gel plate chromatography, boron trifluoride diethyl ether (1.8 mL, 15 mmol) was added, and the mixture was refluxed for 12–15 h to obtain a dark blue mixed solution. The mixture was cooled to room temperature, and the reaction mixture was poured into water and extracted with CH2Cl2. The organic layer was dried with 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). The mixture was refluxed for 3–4 h under anhydrous and oxygen-free conditions at 80 °C and N2 protection. After cooling to room temperature, the solvent was removed by pressure evaporation, followed 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 HRMS mass spectrum of probe compound 1 is shown below. Figure 1 .

[0040] The absorption and fluorescence spectra of probe compound 1 are as follows: Figure 2 and Figure 3 .

[0041] Sensing performance of probe compound 1 for cysteine

[0042] The prepared fluorescent probe compound 1 was dissolved in N,N-dimethylformamide solution, and the sensing performance of compound 1 for cysteine ​​was tested. In the presence of cysteine ​​(10 μM), a decrease in UV-Vis absorption at 675 nm and an increase in UV-Vis absorption at 750 nm were observed after 2 min. Figure 4 As shown. This change in UV-vis absorption is accompanied by a gradual quenching of near-infrared fluorescence emission at 702 nm, as... Figure 5 As 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, glutamic acid, serine, methionine, glutamine, glutathione, etc.) were detected.

[0046] like Figure 6 As shown, the near-infrared fluorescence quenching of compound 1 occurred within 2 minutes only in the presence of cysteine, with minimal fluorescence attenuation caused by other analytes. The new peak at 750 nm generated by compound 1 occurred only in the presence of cysteine, with virtually no effect from other analytes. Figure 7 As 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 as follows: Figure 8As shown, observations revealed that when cells were incubated with only probe compound 1, they emitted weak fluorescence due to the presence of cysteine ​​within the cells. When cells were incubated with both compound 1 and cysteine, the fluorescence was quenched. However, when cells were incubated with both compound 1 and NEM, the fluorescence in HeLa cells was significantly enhanced compared to the previous two groups, as NEM is an effective thiol blocking agent. This indicates that probe compound 1 can be used to detect cysteine ​​in cells.

[0050] In summary, the above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the scope of the technology disclosed in the present invention shall fall within the scope of protection of the present invention.

Claims

1. A near-infrared fluorescent probe, characterized by, having the structure of compound 1: Compound 1.

2. The method of synthesizing the near-infrared fluorescent probe according to claim 1, characterized in that, comprising the following steps: Step 1, adding 1 equivalent of DPP-Br and 2-6 equivalents of 2-amino-3-((2-hexyldecyl)oxy)pyridine into dry toluene, refluxing at 110 °C under N2protection, after fully dissolved, adding 5-10 equivalents of TiCl4into the mixed solution, after 10-15 min of continuous refluxing and stirring, adding 20-25 equivalents of NEt3; continuing refluxing and stirring for 2-3 h, after monitoring the formation of intermediate imine by thin layer chromatography on silica gel plate, adding 22-37 equivalents of BF3•OEt2, then continuing refluxing for 12-15 h; cooling to room temperature, pouring the reaction mixture into water, extracting with CH2Cl2; drying the organic layer with anhydrous sodium sulfate, filtering, removing CH2Cl2by rotary evaporation, obtaining the crude product, which was separated by column chromatography to obtain compound 2; Step 2, adding 1 equivalent of compound 2, 0.2-0.5 equivalents of Pd(dppf)Cl2•CH2Cl2, 15-20 equivalents of KOAc and 3-5 equivalents of 4-formylbenzeneboronic acid into dry DMF, refluxing under anhydrous and oxygen-free, N2protection, cooling to room temperature, evaporating under reduced pressure to remove the solvent, then separating 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 and 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 the TiCl4 is 6 equivalents.

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

6. The method for synthesizing the near-infrared fluorescent probe according to claim 2, characterized in that, The temperature of the reflux in step 2 is 80℃, refluxing for 3-4 h.

7. Use of compound 1 of claim 1 in the preparation of a kit for detecting intracellular cysteine.

8. A kit for detecting cysteine, characterized by, containing the near-infrared fluorescent probe of claim 1 and N-ethylmaleimide.

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