A method for preparing a carbazole-based biothiol near-infrared ratiometric fluorescent probe and its application.
Patent Information
- Application Number
- CN202410616900.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-17
- Publication Date
- 2025-10-28
- Estimated Expiration
- 2044-05-17
AI Technical Summary
[0003]通过相关文献报道，色烯衍生物具有良好的生物硫醇检测位点，但目前报道的荧光探针仍存在一些局限性，如合成路线复杂，单发射波长变化，发射波长短，响应时间长，斯托克斯位移小，不具有线粒体靶向功能等
[0017]1.本发明提供的荧光探针的制备方法简单，可操作性强，原料易得，成本较低。
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Figure CN118745170B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of optical imaging, specifically to a method for preparing a carbazole-based biothiol near-infrared ratiometric fluorescent probe and its application. Background Technology
[0002] Cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are three very important small-molecule thiols that play crucial physiological roles in biological systems. Changes in the intracellular concentrations of these biothiols are closely linked to several diseases. For example, cysteine (Cys) deficiency can lead to slow growth in children, skin lesions, liver damage, edema, narcolepsy, and weakness. Diseases such as Parkinson's disease and Alzheimer's disease are closely associated with high levels of homocysteine (Hcy) in human plasma. Abnormal glutathione (GSH) concentrations may contribute to cancer, neurodegenerative diseases, and HIV / AIDS. Therefore, monitoring biothiols in biological samples is highly valuable.
[0003] According to relevant literature, chromone derivatives possess excellent biothiol detection sites. However, currently reported fluorescent probes still have some limitations, such as complex synthetic routes, single emission wavelength variations, short emission wavelengths, long response times, small Stokes shifts, and lack of mitochondrial targeting function. Therefore, designing and synthesizing novel biothiol fluorescent probes using chromone derivatives as recognition groups is of great significance. Summary of the Invention
[0004] To address the aforementioned problems, this invention provides a novel near-infrared ratiometric fluorescent probe for identifying biothiols and capable of targeted localization to mitochondria. This fluorescent probe has a short reaction time, enabling rapid detection of biothiols, and exhibits high selectivity, sensitivity, and a large Stokes shift. Furthermore, it can be targeted to mitochondria, thereby achieving imaging analysis at specific locations.
[0005] To achieve the above objectives, the present invention employs the following technical solutions:
[0006] A carbazole-based biothiol near-infrared ratiometric fluorescent probe, wherein the carbazole-based biothiol near-infrared ratiometric fluorescent probe is (E)-4-(2-(9-ethyl-9H-carbazol-3-yl)vinyl)-1-((1-oxo-1,2,3,3a-tetrahydrocyclopentan[b]chromium-7-yl)methyl)quinoline-1-ammonium bromide, with the following structural formula:
[0007]
[0008] A method for preparing a carbazole-based biothiol near-infrared ratiometric fluorescent probe includes the following steps:
[0009] Under argon protection, (E)-9-ethyl-3-(2-(quinolin-4-yl)vinyl)-9H-carbazole (compound 2) and 7-(bromomethyl)-3,3a-dihydrocyclopentanthrocyclo-1(2H)-one (compound 3) were dissolved in acetonitrile. The mixture was heated under reflux and stirred until the reactants had reacted completely. The reaction solution was cooled to room temperature, and the solid was precipitated with ethyl acetate. The solid was filtered and washed with ethyl acetate. The crude solid product was collected and subjected to silica gel column chromatography to obtain (E)-4-(2-(9-ethyl-9H-carbazole-3-yl)vinyl)-1-((1-oxo-1,2,3,3a-tetrahydrocyclopentanthrocyclo-7-yl)methyl)quinolin-1-ammonium bromide (compound 1).
[0010]
[0011] Furthermore, the molar ratio of (E)-9-ethyl-3-(2-(quinolin-4-yl)vinyl)-9H-carbazole and 7-(bromomethyl)-3,3a-dihydrocyclopentane[b]chromium-1(2H)-one is 1:1.3 to 2.5.
[0012] Furthermore, the temperature of the stirring reaction is 90℃~100℃, and the stirring reaction time is 3h~14h.
[0013] Furthermore, the reaction is monitored by TLC (Transient Chromatography) until the reactants have completely reacted.
[0014] This route enables the green and efficient synthesis of a carbazole-based biothiol near-infrared ratiometric fluorescent probe (E)-4-(2-(9-ethyl-9H-carbazole-3-yl)vinyl)-1-((1-oxo-1,2,3,3a-tetrahydrocyclopentane[b]chromium-7-yl)methyl)quinoline-1-ammonium bromide.
[0015] Application of a carbazole-based near-infrared ratiometric fluorescent probe for the rapid detection of biothiols, applied to intracellular biothiols and mitochondrial targeted imaging.
[0016] Compared with the prior art, the present invention has the following advantages:
[0017] 1. The method for preparing the fluorescent probe provided by this invention is simple, highly operable, uses readily available raw materials, and has a low cost.
[0018] 2. The fluorescent probe provided by this invention can be used for rapid fluorescence detection of biothiols, and has good selectivity, sensitivity and large Stokes shift.
[0019] 3. The fluorescent probe provided by this invention has good biocompatibility and can be applied to intracellular biothiol imaging.
[0020] 4. This invention utilizes the negative membrane potential of mitochondria to design a positively charged fluorescent probe, which is then located in the mitochondria through electrostatic attraction, thereby achieving targeted imaging of mitochondria.
[0021] 5. The fluorescent probe provided by this invention utilizes the fluorescence intensity ratio (F) at two wavelengths, 530 nm and 650 nm. 530nm / F 650nm This method enables the detection of bio-thiols by changing wavelengths, overcoming the limitations of existing single-wavelength detection methods which have low accuracy. Attached Figure Description
[0022] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the probe of this invention.
[0023] Figure 2 This is the carbon NMR spectrum of the probe of this invention.
[0024] Figure 3 These are the UV and fluorescence spectra of the fluorescent probe of this invention before and after the reaction with biothiols.
[0025] Figure 4 This is the ultraviolet spectrum of 30 μM Cys over time detected by the fluorescent probe of this invention.
[0026] Figure 5 This is the fluorescence spectrum of 30 μM Cys over time detected by the fluorescent probe of this invention.
[0027] Figure 6 The fluorescence ratio (F) of the fluorescent probe of this invention titrated with different Cys concentrations is... 530nm / F 650nm Change diagram.
[0028] Figure 7 The fluorescence ratio (F) of the fluorescent probe of this invention after the addition of biothiols and different amino acids is... 530nm / F 650nm Change diagram.
[0029] Figure 8 This invention relates to the imaging of biothiols in living cells using fluorescent probes.
[0030] Figure 9 This invention provides imaging of the fluorescent probe's targeted localization in mitochondria. Detailed Implementation
[0031] Example 1: Specific synthesis process of the fluorescent probe of the present invention
[0032] (1) The specific synthetic steps of compound 2 are as follows:
[0033]
[0034] N-ethylcarbazole-3-carbaldehyde (compound 7) (2.23 g, 10 mmol), 4-methylquinoline (compound 8) (2.15 g, 15 mmol), trimethylchlorosilane (TMSCl, 10.86 g, 100 mmol), and N,N-dimethylformamide (DMF, 20 mL) were added to a pressure reactor. The reaction mixture was heated to 105 °C and the reaction was sealed for 24 h. After the reaction was completed, the reaction mixture was cooled to room temperature, ultrapure water was added, the mixture was extracted with dichloromethane, the combined organic phases were washed with brine, dried over anhydrous sodium sulfate, and the filtrate was concentrated to give a crude product. This crude product was separated by silica gel column chromatography (ethyl acetate / dichloromethane = 1:5) to give solid product compound 2 (yield 42%), namely (E)-9-ethyl-3-(2-(quinoline-4-yl)vinyl)-9H-carbazole.
[0035] (2) The specific synthetic steps of compound 3 are as follows:
[0036]
[0037] Under argon protection, salicylaldehyde (compound 6) (6.11 g, 50 mmol) was dissolved in concentrated hydrochloric acid (50 mL), and then a 37% formaldehyde aqueous solution (4 mL) was added. The mixture was heated to 70 °C and stirred for 24 h. After the reaction was completed, it was cooled to room temperature, filtered, and the solid was washed with ultrapure water. The solid was collected and dissolved in dimethyl sulfoxide (DMSO, 20 mL) and ultrapure water (10 mL). Then, copper sulfate pentahydrate (12.48 g, 50 mmol) was added, and the mixture was heated to 110 °C and stirred for 8 h. After the reaction was completed, it was cooled to room temperature and extracted with dichloromethane. The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, and the filtrate was concentrated to obtain the crude product. This crude product was separated by silica gel column chromatography (ethyl acetate / petroleum ether = 1:3 to 1:1) to obtain the solid product compound 5 (yield 30%), namely 2-hydroxy-5-hydroxymethylbenzaldehyde.
[0038]
[0039] Under argon protection, 2-hydroxy-5-hydroxymethylbenzaldehyde (compound 5) (1.52 g, 10 mmol), 2-cyclopenten-1-one (1.23 g, 15 mmol), and imidazole (1.02 g, 15 mmol) were dissolved in tetrahydrofuran (THF, 5 mL) and ultrapure water (5 mL). The mixture was stirred at room temperature for 48 h. After the reaction was completed, the mixture was extracted with dichloromethane, and the combined organic phases were washed successively with ultrapure water and brine, dried over anhydrous sodium sulfate, and the filtrate was concentrated to obtain the crude product. This crude product was separated by silica gel column chromatography (ethyl acetate / petroleum ether = 1:3 to 1:1) to obtain the solid product compound 4 (yield 26%), namely 7-(hydroxymethyl)-3,3a-dihydrocyclopentenyl[b]cyclo[b]-1(2H)-one.
[0040]
[0041] Under argon protection, 7-(hydroxymethyl)-3,3a-dihydrocyclopentane[b]chromanthro-1(2H)-one (compound 4) (432.4 mg, 2 mmol) and triphenylphosphine (1.05 g, 4 mmol) were dissolved in dichloromethane (DCM, 20 mL), and then N-bromosuccinimide (NBS, 711.9 mg, 4 mmol) was added. The mixture was stirred at room temperature for 1 h, then washed successively with water and brine, dried over anhydrous sodium sulfate, and the filtrate was concentrated to give a crude product. This crude product was separated by silica gel column chromatography (ethyl acetate / petroleum ether = 1:5 to 1:4) to give solid product compound 3 (yield 40%), namely 7-(bromomethyl)-3,3a-dihydrocyclopentane[b]chromanthro-1(2H)-one.
[0042] Example 2: The specific synthesis steps of the fluorescent probe are as follows:
[0043]
[0044] Under argon protection, (E)-9-ethyl-3-(2-(quinolin-4-yl)vinyl)-9H-carbazole (compound 2) (104.5 mg, 0.3 mmol) and 7-(bromomethyl)-3,3a-dihydrocyclopentane[b]-1(2H)-one (compound 3) (111.6 mg, 0.4 mmol) were dissolved in acetonitrile (4 mL). The reaction mixture was heated to 90 °C and stirred for 8 h. After the reaction was completed, The solid was precipitated with ethyl acetate after cooling to room temperature. The solid was filtered and washed with ethyl acetate. The crude solid product was collected and separated by silica gel column chromatography (dichloromethane / methanol = 20:1) to give solid product compound 1 (yield 94%), namely (E)-4-(2-(9-ethyl-9H-carbazole-3-yl)vinyl)-1-((1-oxo-1,2,3,3a-tetrahydrocyclopentane[b]chromium-7-yl)methyl)quinoline-1-ammonium bromide.1 H NMR(400MHz,d6-DMSO,ppm)δ9.51(d,J=6.8Hz,1H),9.18(d,J=8.0Hz,1H),8.92(s,1H),8.64(d,J=6.8Hz,1H),8.56(d,J=15.6Hz,1H ),8.43-8.39(m,2H),8.28(d,J=8.0Hz,1H),8.20-8.16(m,2H),8.03(d,J=7.6Hz,1H),7.80(d,J=8.8Hz,1H),7.71(d,J=8.4Hz,1H), 7.56-7.52(m,1H),7.47(d,J=2.0Hz,1H),7.42-7.39(m,1H),7.34(t,J=7.6Hz,1H),7.18(d,J=2.0Hz,1H),6.99(d,J=8.4Hz,1H),6. 17(s,2H),5.32-5.27(m,1H),4.55(q,J=7.2Hz,2H),2.63-2.56(m,1H),2.46-2.31(m,2H),2.05-1.95(m,1H),1.38(t,J=7.2Hz,3H). 13 C NMR(100MHz,d6-DMSO,ppm)δ201.09,154.81,153.98,147.49,145.86,141.48 ,140.29,138.01,135.17,132.91,131.29,129.33,128.98,128.25,127.84,12 7.00, 126.77, 126.67, 125.58, 123.00, 122.35, 122.25, 120.74, 119.93, 119.43, 116.83, 116.25, 115.52, 109.92, 75.57, 58.27, 37.39, 36.71, 27.68, 13.89.
[0045] Example 3: UV and fluorescence spectra of the fluorescent probe of the present invention before and after reaction with biothiol.
[0046] After adding 30 μM Cys, GSH, or Hcy to the fluorescent probe solution of this invention (10 μM, PBS:DMSO = 1:1) for 6 min, the changes in UV and fluorescence spectra were measured. The results are shown below. Figure 3 .
[0047] like Figure 3 As shown in (a), the maximum UV absorption peaks of the fluorescent probe solution of the present invention before and after the addition of Cys, GSH, or Hcy are located at 496 nm and 388 nm, respectively. Figure 3 As shown in (b), the fluorescence emission spectrum (458 nm excitation, slit size: 5 nm / 10 nm) blue-shifted from 650 nm to 530 nm. This indicates that the fluorescent probe of this invention reacts with biothiols (Cys, GSH, and Hcy) and can be used to trace biothiols.
[0048] Example 4: Ultraviolet and fluorescence spectra of Cys over time detected by the fluorescent probe of the present invention.
[0049] After adding 30 μM Cys to the fluorescent probe solution of this invention (10 μM, PBS:DMSO = 1:1), the changes in UV and fluorescence spectra over time were measured. The results are shown below. Figure 4 and Figure 5 .
[0050] like Figure 4 As shown in (a), after adding 30 μM Cys to the fluorescent probe solution of the present invention, the absorbance at 496 nm decreased rapidly, while the absorbance at 388 nm gradually increased; from Figure 4 As shown in (b), within 6 minutes, the absorbance at 496 nm reaches its minimum, while the absorbance at 388 nm reaches its maximum. Simultaneously, the fluorescence spectrum also undergoes significant changes, such as... Figure 5 As shown in (a), after adding 30 μM Cys to the fluorescent probe solution of the present invention, the fluorescence intensity at 650 nm rapidly decreases, while the fluorescence intensity at 530 nm gradually increases; from Figure 5 As can be seen in (b), within 6 minutes, the fluorescence ratio (F) 530nm / F 650nm The fluorescence ratio (F) changed significantly and reached its maximum value. This indicates that the fluorescent probe of this invention can be affected by the fluorescence ratio (F). 530nm / F 650nm It enables the detection of biothiols and features a fast response speed and a large Stokes shift (154 nm).
[0051] Example 5: Fluorescence ratio (F) of the fluorescent probe of the present invention titrated with different Cys concentrations 530nm / F 650nm Change diagram.
[0052] Different concentrations of Cys were added to the fluorescent probe solution (10 μM, PBS:DMSO = 1:1) of this invention, and the fluorescence ratio (F) was measured. 530nm / F 650nm (Changes over time.) See results. Figure 6 .
[0053] like Figure 6 As shown, the fluorescence ratio (F) of the fluorescent probe solution of the present invention with the addition of different concentrations of Cys is shown. 530nm / F650nm ) Changes (458nm excitation, gap: 5nm / 10nm). From Figure 6 As can be seen in (a) of the paper, the fluorescence ratio (F) increases with increasing Cys concentration. 530nm / F 650nm Increase larger and faster; from Figure 6 In (b), it can be found that the fluorescence ratio (F) 530nm / F 650nm The good linearity between the fluorescent probe and Cys in the low concentration range indicates that this fluorescent probe has high sensitivity.
[0054] Example 6: Fluorescence ratio (F) of the fluorescent probe of the present invention after the addition of biothiols and different amino acids. 530nm / F 650nm (Change diagram)
[0055] The fluorescent probe solution of this invention (10 μM, PBS:DMSO = 1:1) was supplemented with 30 μM of cysteine (Cys), glutathione (GSH), homocysteine (Hcy), alanine (Ala), arginine (Arg), aspartic acid (Asp), glutamine (Gln), glutamate (Glu), histidine (His), leucine (Leu), lysine (Lys), proline (Pro), and serine (Ser), respectively. The fluorescence spectra were measured after 6 min. The results are shown below. Figure 7 .
[0056] like Figure 7 As shown, only biothiols (Cys, GSH, and Hcy) cause fluorescence ratio (F) 530nm / F 650nm The concentration of β-amino acids increased significantly, while other amino acids remained almost unchanged. This indicates that the fluorescent probe of this invention has good selectivity for biothiols (Cys, GSH, and Hcy), and can specifically recognize biothiols to a certain extent, providing a good foundation for bioimaging.
[0057] Example 7: Imaging of biothiols in living cells using the fluorescent probe of the present invention.
[0058] HeLa cells were cultured in a low-glucose medium containing 10% fetal bovine serum at 37°C in a 5% CO2 saturated humidity incubator. The medium was changed every 2-3 days, and the cells were passaged and transferred to confocal dishes for further culture. Two confocal dishes were used. One dish was directly incubated with 5 μM of the fluorescent probe of this invention for 30 min before imaging. The other dish was first incubated with 1 mM of the thiol scavenger N-ethylmaleimide (NEM) for 30 min, washed with phosphate-buffered saline (PBS), and then incubated with 5 μM of the fluorescent probe of this invention for 30 min before imaging. The excitation wavelength was 458 nm, the green window fluorescence collection wavelength was 480-550 nm, and the red window fluorescence collection wavelength was 620-690 nm. The results are shown below. Figure 8 .
[0059] from Figure 8 It can be observed that cells treated solely with the fluorescent probe of this invention exhibit stronger fluorescence in the green window than in the red window, with a larger fluorescence intensity ratio (green window / red window). Conversely, cells treated first with NEM and then with the fluorescent probe of this invention show weaker fluorescence in the green window than in the red window, with a smaller fluorescence intensity ratio (green window / red window). This indicates that the fluorescent probe can ratiometrically detect changes in intracellular biothiol concentrations.
[0060] Example 8: Imaging of the fluorescent probe of the present invention targeting and localizing mitochondria.
[0061] HeLa cells were cultured in a low-glucose medium containing 10% fetal bovine serum at 37°C in a 5% CO2 saturated humidity incubator. The medium was changed every 2-3 days, and the cells were passaged and transferred to confocal dishes for further culture. In one confocal dish, the medium was replaced with 1 mL of fresh medium. The dish was incubated with 1 mM of the thiol scavenger N-ethylmaleimide (NEM) for 30 min, washed with phosphate-buffered saline (PBS), and then incubated with 5 μM of the fluorescent probe of this invention and 500 nM of the commercially available mitochondrial-targeting fluorescent probe Mito-Tracker Green for another 30 min before imaging. The green window excitation wavelength of the fluorescent probe of this invention was 458 nm, and the fluorescence collection wavelength was 620-690 nm; the red window excitation wavelength of Mito-Tracker Green was 488 nm, and the fluorescence collection wavelength was 495-530 nm. Results are shown below. Figure 9 .
[0062] from Figure 9 It can be observed that the fluorescent probe of the present invention has good overlap with commercial mitochondrial-targeting fluorescent probes, indicating that the fluorescent probe of the present invention is located in the mitochondria of cells.
[0063] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. For those skilled in the art, several modifications and improvements can be made without departing from the principle of the present invention, and all of them are included within the protection scope of the present invention.
[0064] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.
Claims
1. A carbazole-based biothiol near-infrared ratiometric fluorescent probe, characterized in that, The carbazole-based biothiol near-infrared ratiometric fluorescent probe is (E)-4-(2-(9-ethyl-9H-carbazol-3-yl)vinyl)-1-((1-oxo-1,2,3,3a-tetrahydrocyclopentanol[b]chromium-7-yl)methyl)quinoline-1-ammonium bromide, with the following structural formula:
2. A method for preparing a carbazole-based biothiol near-infrared ratiometric fluorescent probe, characterized in that, Includes the following steps: Under argon protection, (E)-9-ethyl-3-(2-(quinolin-4-yl)vinyl)-9H-carbazole and 7-(bromomethyl)-3,3a-dihydrocyclopentanion-1(2H)-one were dissolved in acetonitrile. The mixture was heated under reflux and stirred until the reactants had reacted completely. The reaction solution was cooled to room temperature, and the solid was precipitated with ethyl acetate. The solid was filtered and washed with ethyl acetate. The crude solid product was collected and subjected to silica gel column chromatography to obtain (E)-4-(2-(9-ethyl-9H-carbazole-3-yl)vinyl)-1-((1-oxo-1,2,3,3a-tetrahydrocyclopentanion-7-yl)methyl)quinolin-1-ammonium bromide.
3. The method for preparing a carbazole-based biothiol near-infrared ratiometric fluorescent probe according to claim 2, characterized in that, The molar ratio of (E)-9-ethyl-3-(2-(quinolin-4-yl)vinyl)-9H-carbazole to 7-(bromomethyl)-3,3a-dihydrocyclopentane[b]chromium-1(2H)-one is 1:1.3 to 2.
5.
4. The method for preparing a carbazole-based biothiol near-infrared ratiometric fluorescent probe according to claim 2, characterized in that, The temperature of the stirring reaction is 90℃~100℃, and the stirring reaction time is 3h~14h.
5. The method for preparing a carbazole-based biothiol near-infrared ratiometric fluorescent probe according to claim 2, characterized in that, The reaction was monitored by TLC plate spotting until the reactants had completely reacted.
6. The application of the carbazole-based biothiol near-infrared ratiometric fluorescent probe as described in claim 1, characterized in that, The near-infrared ratiometric fluorescent probe is used to prepare reagents for rapid detection of biothiols, and is applied to the preparation of reagents for intracellular biothiols imaging and targeting to mitochondria.