Preparation and application of peroxynitroso fluorescent probe based on thioxanthene
By designing a fluorescent probe based on thioxanthene, the problem of spontaneous fluorescence interference of fluorescent probes in the detection of peroxynitrite in the existing technology is solved, and highly sensitive and selective near-infrared detection is achieved, which is suitable for the detection of peroxynitrite in complex biological systems.
Patent Information
- Application Number
- CN202510740387.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-04
- Publication Date
- 2025-09-16
AI Technical Summary
Existing fluorescent probes are easily interfered by endogenous autofluorescence when detecting peroxynitrite (ONOO-) and lack long-wavelength emission capability, which limits their application in complex biological systems.
A thioanthracene-based fluorescent probe was designed and synthesized. By replacing oxygen atoms with sulfur atoms, the absorption and emission wavelengths were extended, and a fluorescent probe capable of high-sensitivity detection in the near-infrared region was developed.
It achieves highly sensitive detection of peroxynitrite in complex biological systems, has good selectivity and response speed, and can accurately track and detect peroxynitrite levels in cells under physiological conditions.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to the preparation and application of a peroxynitroso fluorescent probe based on thioxanthene. Background Art
[0002] Peroxynitrite (ONOO - ) is an intermediate formed in vivo by the reaction of superoxide and nitric oxide free radicals (JA Miles, JL Egan, JA Fowler, P. Machattou, AD Millard, CJ Perry, DJ S Canlan, PC Taylor, Biochem. Biophys. Res. Commun., 2021, 580: 107-112). As a strong oxidant and strong nucleophile, ONOO - It can react with a variety of biomolecules, such as proteins, lipids, nucleic acids, etc., ultimately leading to cell death (L. Liaudet, G. Vassalli, P. Pacher, Front. Biosci. Biotechnol. Biochem., 2009, 14: 4809-4814). - It can also act as a signaling molecule and participate in signal transduction processes (BC Dickinson, CJ Chang, Nat. Chem. Biol., 2011, 7: 504-511). In addition, abnormal levels of peroxynitrite are associated with some common diseases, such as cardiovascular disease, neurological diseases such as Parkinson's disease, diabetes and inflammatory bowel disease, epilepsy, etc. (YX Guo, HW Huang, Q. Zhang, HJ Wang, MM Liu, Anal. Chim. Acta, 2025, 1350: 343863; M. Yan, HX Fang, XQ Wang, JJ Xu, CW Zhang, L. Xu, L. Li, Sensor Actuat. B Chem., 2021, 328: 129003; JZ Hua, Tadeusz Malinski, Int. J. Nanomed., 2019, 8973-8987). Because nitrite peroxide has important physiological and clinical significance, it is crucial to design effective methods to achieve its accurate detection in complex biological systems.
[0003] In recent years, fluorescent probes have attracted widespread attention due to their advantages such as simple operation, high sensitivity, non-invasive detection characteristics and high spatiotemporal resolution (H. Zhu, JL Fan, JJ Du, X. Peng, Acc. Chem. Res., 2016, 49: 2115-2126). - Due to its strong oxidizing and nucleophilic properties, many fluorescent probes have been developed to detect ONOO - (ZF Ma, H. Chen, ZJ Xia, J. You, C C Han, Int. J. Mol. Sci., 2023, 24: 12821; SJ, CX, LW, Theranostics, 2023, 135: 1716; Y. Li, X. Xie, X. Yang, M. Li, X. Jiao, Y. Sun, X. Wang, B. Tang, Chem. Sci., 2017, 8: 4006-4011). However, the emission wavelengths of the fluorophores of these probes (such as fluorescein, naphthalimide, fluoroborane, xanthene, etc.) are relatively short, and in practical applications, they often face interference from endogenous autofluorescence, which seriously limits their in-depth application in complex biological systems. Therefore, it is very meaningful to design and synthesize near-infrared fluorescent probes with long-wavelength emission that can achieve high-sensitivity detection of peroxynitrite.
[0004] Compared with traditional xanthene fluorescent probes, thioxanthene uses a strategy of replacing oxygen atoms with sulfur atoms to prolong absorption and emission, which is beneficial to improving tissue penetration and anti-autofluorescence interference properties. Fluorescent probes based on thioxanthene dyes have been successfully used to detect some targets, such as cysteine (Cys), nitroreductase (NTR), etc. (L.Li, Z.Zhang, L.Zhou, Anal.Chem. 2024, 96, 7248-7256; C.Hu, H.Liu, Z.Zhang, L.Li, GJMao, GJCheng, Small, 2025, 21: 2570156). However, to date, there has been no research using thioxanthene dyes as fluorescent probes to detect ONOO. - Therefore, a thioxanthene dye-based fluorescent probe was designed and synthesized to detect ONOO - It is very necessary. Summary of the Invention
[0005] According to the requirements put forward, the present inventors have conducted in-depth research on this and, after paying a lot of creative work, have provided a peroxynitrite fluorescent probe based on thioxanthene.
[0006] The technical solution of the present invention is a peroxynitroso fluorescent probe based on thioxanthene, the structure of which is as follows:
[0007]
[0008] A method for preparing a peroxynitroso fluorescent probe based on thioxanthene, comprising the following steps:
[0009] Synthesis of compound SNR: 1.0 equivalent of SNR-OH, 1.0 to 3.0 equivalents of 4-(bromomethyl)phenylboronic acid pinacol ester, and 1.0 to 2.0 equivalents of anhydrous potassium carbonate are added sequentially to a 100 mL round-bottom flask, and then 5 to 10 mL of anhydrous N,N-dimethylformamide is added and dissolved. Under nitrogen protection, the mixture is heated to 80° C. and stirred for 4 to 8 hours. After the reaction is completed, the crude product is freed from the solvent under reduced pressure and purified by column chromatography using a CH2Cl2 / CH3OH eluent with a volume ratio of 200:1 to 50:1 to obtain a blue solid compound SNR, which is the fluorescent probe.
[0010] The beneficial effect of the present invention is that a peroxynitroso fluorescent probe based on thioxanthene has good performance. First, the SNR of ONOO - Absorption spectrum in the presence of. SNR has an obvious absorption peak at 630nm. When ONOO is added - After that, the absorption peak at 630nm disappeared, and a new absorption peak appeared at 740nm. After comparison, it was found that the peak shape of this new absorption peak was consistent with the peak shape of the fluorophore SNR-OH. - The fluorescence response of ONOO is almost non-signal. - As the concentration increased, the fluorescence emission intensity at 785nm increased significantly. Further analysis showed that in the presence of ONOO - The fluorescence intensity at 785 nm and ONOO - The concentration showed a good linear relationship. The detection limit of the detection system (calculated according to the formula 3σ / k) was 0.03μM. These results show that SNR can be used for high-sensitivity ONOO - Then, the selectivity of the probe was studied and the selectivity of SNR was evaluated by measuring the fluorescence response of SNR to reactive oxygen species, reactive nitrogen species, reactive sulfur species and biothiols. The results showed that SNR can only be detected by ONOO - The fluorescence intensity of ONOO is turned on by SNR, while the effect of other analytes on the fluorescence intensity is almost negligible, indicating that SNR can be used to detect ONOO in complex biological systems. - Finally, the effect of pH on the fluorescence probe determination of ONOO- When the pH value is between 7.4 and 8.0, it does not affect the fluorescence probe's response to ONOO - In addition, the fluorescent probe has a fast response time of less than 10s.
[0011] An application of a thioxanthene-based peroxynitroso fluorescent probe. RAW264.7 cells were used for research. After adding the fluorescent probe to the cells, no obvious fluorescence change was produced, indicating that in normal cells, ONOO - The content of ONOO is maintained at a low level. SIN-1 is added to the cells to increase exogenous ONOO - After that, a strong fluorescence can be clearly observed. In addition, lipopolysaccharide (LPS) was used to induce cell inflammation, and a significant increase in cell fluorescence intensity was also observed. In the inflammatory cells induced by LPS, uric acid (UA) was added to clear ONOO in the cells. - , then the probe was added, and the fluorescence in the cells was found to be significantly weakened. These results indicate that SNR can effectively inhibit the expression of exogenous and endogenous ONOO in RAW264.7 cells. - It has tracking and detection capabilities, which provides a reliable means to monitor peroxynitrite levels in living cells. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 The synthetic route of fluorescent probe.
[0013] Figure 2 As a fluorescent probe with different concentrations of ONOO - Fluorescence spectrum after action.
[0014] The horizontal axis is the wavelength, and the vertical axis is the fluorescence intensity. The concentration of the fluorescent probe is 10 μM, ONOO - The concentrations are: 0, 0.1, 0.5, 1, 2, 4, 6, 8, 10, 12, 14 μM. The emission wavelength is 750-900 nm, and the corresponding excitation wavelength is 700 nm.
[0015] Figure 3 As fluorescent probes for different ONOO - The fluorescence linear response plot of the concentration.
[0016] Figure 4 As a fluorescent probe with ONOO - UV-visible absorption spectrum after action.
[0017] The horizontal axis is wavelength, and the vertical axis is absorbance. The concentration of the fluorescent probe is 10 μM, ONOO - The concentration is 14 μM.
[0018] Figure 5Figure 2 is the selectivity diagram of the fluorescent probe.
[0019] The concentration of fluorescent probes was 10 μM, ONOO - The concentration was 14 μM, and the concentrations of other analytes were all 200 μM.
[0020] Figure 6 This is a diagram showing the effect of pH on the fluorescent probe.
[0021] Figure 7 As a fluorescent probe with ONOO - The relationship between fluorescence intensity and time after the action.
[0022] Figure 8 This is a cytotoxicity test. The horizontal axis is the concentration of the fluorescent probe, and the vertical axis is the cell survival rate.
[0023] Figure 9 Fluorescent probes and ONOO - Cell imaging of the effect.
[0024] Control group: Cells were incubated with the probe for 30 minutes. SIN-1 group: Cells were first treated with SIN-1 for 60 minutes, followed by SNR for 30 minutes. LPS group: Cells were first treated with SIN-1 for 12 hours, followed by SNR for 30 minutes. LPS+UA group: Cells were treated with SIN-1 and UA for 12 hours, respectively, followed by SNR for 30 minutes.
[0025] Figure 10 The relative fluorescence intensity of cells. DETAILED DESCRIPTION
[0026] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but is not limited thereto.
[0027] Example 1:
[0028] Synthesis of fluorescent probes
[0029] Synthesis route such as Figure 1 1.0 equivalent of SNR-OH (162 mg, 0.40 mmol), 2.0 equivalents of 4-(bromomethyl)phenylboronic acid pinacol ester (236 mg, 0.80 mmol), and 1.0 equivalent of anhydrous potassium carbonate (55.2 mg, 0.40 mmol) were added sequentially to a 100 mL round-bottom flask. 5 mL of anhydrous N,N-dimethylformamide was then added and dissolved. Under nitrogen protection, the mixture was heated to 80°C and stirred for 6 h. After the reaction was completed, the crude product was freed from the solvent under reduced pressure and purified by column chromatography using a CH2Cl2 / CH3OH eluent with a volume ratio of 50:1 to obtain a blue solid compound SNR, which is the fluorescent probe.1 HNMR(400MHz,DMSO-d6)δ8.26(d,J=8.0Hz,1H),8.10(d,J=12.0Hz,1H),7.94(d ,J=7.6Hz,1H),7.75(t,J=14.6Hz,1H),7.62-7.69(m,3H),7.42(d,J=8.0Hz,3H) ,7.16(s,1H),7.08(d,J=11.4Hz,1H),7.03(s,1H),6.96-6.99(m,1H),4.73-4.7 8(m,2H),2.60-2.65(m,4H),1.37(t,J=16.0Hz,3H),1.26(s,12H),1.22(s,2H).
[0030] Example 2:
[0031] Fluorescent probes and ONOO - Solution preparation
[0032] Preparation of probe solution: SNR was dissolved in dimethyl sulfoxide (DMSO) to prepare 1.0×10 -4 M probe solution. ONOO was prepared using four solutions of H2O2 (0.7 M), HCl (0.6 M), NaNO2 (0.6 M) and NaOH (1.5 M). - Solution of ONOO - To determine the concentration, it is necessary to measure the absorbance A of the solution at 302nm. The calculation formula is: C ONOO - =A / 1.67 (mM). The absorbance at 302 nm was measured, and the molar extinction coefficient was 1670M -1 cm -1 . Use SNR stock solution and appropriate volume of ONOO - Prepare a series of SNR and ONOO stock solutions - The test solution was then diluted to 5 mL with phosphate buffer solution (PBS, 50 mM, pH 7.4).
[0033] Example 3:
[0034] Fluorescent probes and ONOO - Determination of fluorescence spectrum of the effect
[0035] Figure 2 As a fluorescent probe with ONOO - The fluorescence spectrum of the fluorescent probe is 10 μM, ONOO -The concentrations were 0, 0.1, 0.5, 1, 2, 4, 6, 8, 10, 12, and 14 μM, respectively. The excitation wavelength used in the experiment was 700 nm, and the emission wavelength range was 750–900 nm. The slit width was 5.0 nm / 5.0 nm, and the fluorescence measurement instrument used was a Hitachi F4600 fluorescence spectrophotometer. Figure 3 For probes of different ONOO - The linear response graph of the fluorescence intensity and ONOO - The concentration of ONOO showed a linear relationship, the linear range was 0.1μM~14.0μM, and the detection limit was 0.03μM. This shows that the probe can detect ONOO with high sensitivity. - .
[0036] Example 4:
[0037] Fluorescent probes and ONOO - Determination of UV-visible absorption spectrum of
[0038] Figure 4 As a fluorescent probe with ONOO - UV-visible absorption spectrum after action, the concentration of fluorescent probe is 10μM, ONOO - The concentration of ONOO was 14 μM. The instrument used for UV-visible absorption spectrum measurement was Agilent Cary60 UV-visible spectrophotometer. SNR has a clear absorption peak at 630 nm. - After that, the original absorption peak at 630nm disappeared, and a new absorption peak appeared at 740nm.
[0039] Example 5:
[0040] Fluorescent probe for ONOO - Assay selectivity
[0041] Figure 5 As a fluorescent probe for ONOO - The selectivity of the assay was determined by measuring the SNR for active oxygen (H2O2, ClO - , 1 O2, ·OH), active nitrogen (NO, NO2 - , NO3 - ), active sulfur (H2S, SO3 2- , HSO3 - ) and biothiols (Cys, Hcy, GSH) fluorescence responses to evaluate the selectivity of SNR. The results showed that SNR can only be generated by ONOO - The fluorescence intensity of ONOO is turned on by SNR, while the effect of other analytes on the fluorescence intensity is negligible, indicating that SNR can be used to detect ONOO in complex biological systems. - .from Figure 5It can be seen that only ONOO - It can cause changes in the fluorescence spectrum, and other detection substances have no obvious effect on the fluorescence spectrum of the probe. These results show that the fluorescent probe has a strong effect on ONOO - Have better selectivity.
[0042] Example 6:
[0043] Effect of solution pH on the determination of ONOO by fluorescent probe - The fluorescence properties of
[0044] Study on the effect of pH value on the determination of ONOO by fluorescent probe - The fluorescence spectrum of Figure 6 In order to verify the applicable environment of the probe, the effect of pH value on the fluorescence performance of the SNR probe was evaluated. In the pH range of 4.0-10.0, the SNR probe itself hardly produces fluorescence. When ONOO - After that, a significant fluorescence enhancement was observed in the pH range of 7.4-8.0, and the response signal reached the best state at the physiological pH value (7.4). This feature clearly proves that the SNR probe can achieve ONOO under physiological conditions (pH 7.4). - effective detection.
[0045] Example 7:
[0046] Fluorescent probes and ONOO - Determination of response time of action
[0047] To test the SNR of ONOO - The response time of the probe was analyzed by real-time monitoring of the changes in the 785nm near-infrared fluorescence signal. - The signal peak is reached quickly within 10 seconds.
[0048] Example 8:
[0049] Application of fluorescent probes in living cells
[0050] First, cytotoxicity assays were performed, e.g. Figure 8 When 0-40.0 μM SNR probe was added, the cell survival rate was above 90%, which shows that this fluorescent probe has low toxicity and is expected to be used for detecting ONOO in living cells. - Then, we studied the application of fluorescent probes in living cells and selected RAW264.7 cells for confocal microscopy imaging. The results are as follows: Figure 9 、 Figure 10As shown. In the control group, almost no fluorescence was observed. Then, after adding SIN-1 to the cells and incubating with the probe, a strong fluorescence signal was observed. When LPS was added to the cells, the cells were stimulated to produce endogenous ONOO - After that, the fluorescence was significantly enhanced when the probe was used for treatment. - After adding the probe, the fluorescence in the cells disappeared. These results show that the probe can detect ONOO in cells with high sensitivity. - .
Claims
1. A thioxanthene-based peroxynitrite fluorescent probe, characterized in that: The structure is as follows:
2. The method for preparing the peroxynitroso fluorescent probe of thioxanthene according to claim 1, wherein The reaction steps are as follows: 1.0 equivalent of SNR-OH, 1.0 to 3.0 equivalents of 4-(bromomethyl)phenylboronic acid pinacol ester, and 1.0 to 2.0 equivalents of anhydrous potassium carbonate are sequentially added to a 100 mL round-bottom flask. Then, 5 to 10 mL of anhydrous N,N-dimethylformamide is added and dissolved. Under nitrogen protection, the mixture is heated to 80° C. and stirred for 4 to 8 hours. After the reaction is completed, the crude product is freed from the solvent under reduced pressure and purified by column chromatography using a CH2Cl2 / CH3OH eluent with a volume ratio of 200:1 to 50:1 to obtain a blue solid compound SNR, which is the fluorescent probe.
3. The use of a peroxynitrite fluorescent probe according to claim 1, characterized in that: The fluorescent probe has been applied to cell imaging research and can detect changes in the peroxynitrite content in cells.