Preparation and application of thiazolinone-xanthene peroxynitrite fluorescent probe

Abstract

This invention relates to the preparation and application of a thiazolinone-oxanthracene peroxynitroso fluorescent probe. The probe has the following structural formula: This invention provides a method for synthesizing this fluorescent probe using thiazolinone-oxanthracene dye, pinacol ester of 4-bromomethylphenylboronic acid, etc., as raw materials; this fluorescent probe is a peroxynitroso fluorescent probe with near-infrared emission; firstly, this fluorescent probe is effective against ONOO... ‑ It exhibited good sensitivity, with a 7.5-fold increase in fluorescence intensity; secondly, this fluorescent probe showed good sensitivity to ONOO. ‑ It exhibits high selectivity, unaffected by other reactive oxygen species, biothiols, and amino acids; furthermore, this fluorescent probe is compatible with ONOO. ‑ It acts rapidly, with a response time of less than 5 minutes; in addition, the fluorescent probe is used to detect the content of peroxide nitrosamines in living cells.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to the preparation and application of fluorescent probes based on thiazolinone-oxanthracene peroxynitroso. Background Technology

[0002] peroxynitroso (ONOO) - Reactive oxygen species (ROS) are produced by the diffusion-controlled combination of nitric oxide and superoxide radical anions, and are important reactive oxygen species in living systems (C. Szabó, H. Ischiropoulos, R. Radi, Nat. Rev. Drug Discovery, 2007, 6, 662-680). - As an endogenous reactive oxygen species, it participates in many important physiological and pathological processes (Z. Wang, W. Wang, P. Wang, X. Song, Z. Mao, Z. Liu, Anal. Chem. 2021, 93, 3035-3041). Studies have shown that ONOO - It can react with a large number of biological macromolecules such as proteins and nucleic acids, disrupting their biological functions (H. Kawasaki, K. Ikeda, A. Shigenaga, T. Baba, K. Takamori, H. Ogawa, F. Yamakura, Free Radic. Biol. Med, 2011, 50, 419-427). Simultaneously, ONOO in the body... - Abnormal levels of nitrosamines are considered closely associated with many diseases, such as cardiovascular and cerebrovascular diseases, Alzheimer's disease, inflammation, and cancer (GYLiou, P. Storz, Free Radic. Res, 2010, 44, 479-496; L. Myatt, W. Kossenjans, R. Sahay, A. Eis, J. Matern. Med, 2000, 9, 79-82; S. Feng, D. Liu, G. Feng, Anal. Chim. Acta, 2019, 1054, 137–144; D. Liu, S. Feng, G. Feng, Sens. Actuators B Chem, 2018, 269, 15-21). Given the important physiological and clinical significance of nitrosamines, there is an urgent need to design effective methods to accurately detect their levels.

[0003] Traditional methods for detecting nitrosamine peroxide include colorimetry, electrochemical analysis, gas chromatography, and differential optical absorption spectroscopy. However, these methods typically require complex procedures, lack sufficient sensitivity, and cannot achieve in vivo detection of ONOO. -Fluorescence detection methods have attracted much attention due to their advantages such as high sensitivity, good selectivity, and applicability to bioimaging (Z.Li, J.Li, D.Zhang, X.Zhu, Y.Ye, Y.Zhao, Sens. Actuators B Chem, 2020, 312, 127-944; H.Niu, K.Chen, J.Xu, X.Zhu, W.Cao, Z.Wang, Y.Ye, Y.Zhao, Sens. Actuators BChem, 2019, 299, 126-938). In recent years, many methods for detecting ONOO have been developed. - Fluorescent probes are used for real-time monitoring of ONOO within cells. - Activity. (C. Zhao, J. An, L. Zhou, Q. Fei, F. Wang, J. Tan, B. Shi, R. Wang, Z. Guo and W.-H. Zhu, Chem. Commun., 2016, 52, 2075–2078; Z.-N. Sun, H.-L. Wang, F.-Q. Liu, Y. Chen, P. K. T. Amand D. Yang, Org. Lett., 2009, 11, 1887–1890; J. Miao, Y. Huo, H. Shi, J. Fang, J. Wang and W. Guo, J. Mater. Chem. B, 2018, 6, 4466–4473; T. Peng, X. Chen, L. Gao, T. Zhang, W. Wang, J. Shen and D. Yang, Chem. Sci., 2016, 7, 5407–5413; ZK Wang, L. Wu, YWWang, M. Zhang, ZY Zhao, CY Liu, QX Duan, P. Jia, BC Zhu, Anal. Chim. Acta. 2019, 1049, 219–225; J. Cui, SP Zang, HL Nie, TJShen, S. Su, J. Jing, XL Zhang, Sens. Actuators B Chem., 2021, 328, 129069.). However, these fluorescent probes have relatively short analytical wavelengths and high signal-to-noise ratios, making them susceptible to interference from autofluorescence signals generated by biomolecules in vivo, thus reducing their sensitivity and limiting their application in biological systems. Therefore, designing novel fluorescent probes with near-infrared emission is crucial.

[0004] Thiazolinone-oxanthracene, as a novel fluorescent dye, exhibits near-infrared emission. Therefore, it holds promise for achieving deeper tissue penetration with less interference from autofluorescence, making it more advantageous for bioimaging. Studies have shown that fluorescent probes using oxanthracene have been successfully applied to detect several targets, such as alkaline phosphatase and H2S (L.Xu,X.He,Y.Huang,P.Ma,Y.Jiang,X.Liu,S.Tao,Y.Sun,D.Song andX.Wang,J.Mater.Chem.B,2019,7,1284–1291;X.Zhang,R.Sun,G.Duan,Z.Zhou,Y.Luo,W.Li,L.Zhang,Y.Gu andX.Zha,NewJ.Chem.2018,42,19795-19800). However, to date, there is no use of thiazolinone-oxanthracene dye as a fluorescent probe for the detection of ONOO. - Therefore, a fluorescent probe based on thiazolinone-oxanthracene dye was designed and synthesized to detect ONOO. - It is absolutely necessary. Summary of the Invention

[0005] Based on the requirements, the inventors conducted in-depth research and, after a great deal of creative work, provided a fluorescent probe based on thiazolinone-oxanthracene peroxynitroso.

[0006] The technical solution of this invention is a thiazolinone-oxanthracene peroxynitroso fluorescent probe, namely TX-P, characterized in that its structure is as follows:

[0007]

[0008] A method for preparing a fluorescent probe based on thiazolinone-oxanthracene peroxynitroso, characterized by the following reaction steps:

[0009] Under N2 protection, 1 equivalent of TX-OH, 2.0–2.5 equivalents of 4-bromomethylphenylboronic acid pinacol ester, and 2.0–3.0 equivalents of potassium carbonate were sequentially added to a 50 mL round-bottom flask, followed by the addition of 10–15 mL of DMF to dissolve the TX-OH. The mixture was stirred at 40–70 °C for 5–8 hours. After the reaction was complete, the mixture was extracted with CH2Cl2, and the organic phase was collected. The organic phase was dried with anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography with CH2Cl2 / CH3OH = 80:1–120:1 as the eluent, yielding a dark purple solid product TX-P, which is the fluorescent probe.

[0010] The beneficial effect of this invention is the excellent spectral response performance of a thiazolinone-oxanthracene peroxynitroso fluorescent probe. First, the fluorescence spectral properties of this probe were studied. The probe itself does not have a significant near-infrared emission peak at 725 nm; when ONOO is added to the probe... - Subsequently, a distinct near-infrared emission peak appeared at 725 nm. Furthermore, with ONOO... - With increasing concentration, the near-infrared fluorescence intensity of the probe continuously increases. When 30 μM ONOO is added... - At that time, the fluorescence intensity increased by approximately 7.5 times, thus enabling excellent detection of ONOO. - The probe has a detection range from 2.5 μM to 30 μM and a detection limit of 0.78 μM, indicating that it can detect ONOO with high sensitivity. - Next, the ultraviolet absorption spectrum of the probe was studied. Without the addition of ONOO... - At that time, the probe itself had no ultraviolet absorption peak; when ONOO was added... - After the addition of ATP, the probe exhibited an absorption peak at 650 nm. Then, the selectivity of the probe was investigated, examining its interactions with various metal ions (Na+, Na ... + ,K + Ca 2+ Cu 2+ ) and anions (Cl - ,Br - ,I - NO3 - CO3 2- SO4 2- Biothiols (Cys, Hcy, GSH), amino acids (Lys, Phe, Leu, Val, Trp, Lie, Met, Thr), reactive oxygen species (ClO) - H2O2) and the detectable substance (ONOO) - The fluorescence response of ONOO was examined. The results showed that only ONOO... - The pH value can cause changes in the fluorescence spectrum, while other analytes have no significant effect on the fluorescence spectrum of the probe. Finally, the effect of pH value on the determination of ONOO by the fluorescent probe was investigated. - The effect of pH values ​​between 7.0 and 10.0 does not affect the fluorescence probe's response to ONOO. - The determination was performed. Furthermore, the fluorescent probe responded rapidly, with a response time of less than 5 minutes.

[0011] An application of a thiazolinone-oxanthracene peroxynitroso fluorescent probe. When the fluorescent probe was added to cells, no obvious fluorescence was observed, indicating that ONOO in the cells... -The content was low. Cells treated with lipopolysaccharide (LPS) and interferon-γ (IFN-γ), followed by probe staining, showed strong fluorescence; however, treatment with aminoguanidine hydrochloride (AG) inhibited intracellular ONOO. - The production of ONOO was observed, and intracellular fluorescence was significantly reduced. These results indicate that fluorescent probes can detect ONOO produced intracellularly. - This provides a reliable means of monitoring and treating lesions related to peroxynitrosamines. Attached Figure Description

[0012] Figure 1 This is the synthetic route for the fluorescent probe.

[0013] Figure 2 Fluorescent probes with different concentrations of ONOO - Fluorescence spectrum after treatment.

[0014] The horizontal axis represents wavelength, and the vertical axis represents fluorescence intensity. The concentration of the fluorescent probe is 10 μM, ONOO. - The concentrations were 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, and 30 μM, respectively. The fluorescence excitation wavelength was 650 nm.

[0015] Figure 3 For fluorescent probes to different ONOO - Fluorescence linear response plot for concentration.

[0016] Figure 4 For fluorescent probes and ONOO - The UV-Vis absorption spectrum after the treatment.

[0017] The horizontal axis represents wavelength, and the vertical axis represents absorbance. The concentration of the fluorescent probe is 10 μM, ONOO. - The concentration is 30 μM.

[0018] Figure 5 This is a selectivity diagram of the fluorescent probe.

[0019] The concentration of the fluorescent probe is 10 μM, ONOO - The concentration was 30 μM, and the concentrations of other analytes were all 200 μM.

[0020] Figure 6 This is a graph showing the effect of pH on fluorescent probes.

[0021] Figure 7 For fluorescent probes and ONOO - The relationship between fluorescence intensity and time after treatment is shown in the graph.

[0022] Figure 8This is a graph from a cytotoxicity assay. The horizontal axis represents the concentration of the fluorescent probe, and the vertical axis represents cell viability.

[0023] Figure 9 Fluorescent probes and ONOO - Cellular imaging of the effects. (a) Cells stained with probe for 0.5 h. (b) Cells treated with LPS and IFN-γ for 10 h, then stained with probe for 0.5 h. (c) Cells treated with LPS, IFN-γ, and AG for 10 h, then stained with probe for 0.5 h. Detailed Implementation

[0024] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but is not limited thereto.

[0025] Example 1:

[0026] Synthesis of fluorescent probes

[0027] Synthetic routes such as Figure 1 Under N2 protection, TX-OH (90 mg, 0.2 mmol), pinacol 4-bromomethylphenylboronic acid (118 mg, 0.4 mmol), and potassium carbonate (110 mg, 0.4 mmol) were added sequentially to a 50 mL round-bottom flask, followed by the addition of 15 mL of DMF to dissolve them. The mixture was stirred at 60 °C for 6 hours. After the reaction was complete, the mixture was extracted with CH2Cl2, and the organic phase was collected. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography with CH2Cl2 / CH3OH = 100:1 as the eluent, yielding a dark purple solid product TX-P (76.5 mg, yield 57%), which is the fluorescent probe. 1 H NMR (400MHz, CDCl3): δ8.46(s,1H),7.85(d,J=7.8Hz,2H),7.75-7.72(m,3H),7.62(d,J=7.8Hz,2H),7.43(d,J=7.8Hz,2H),7.38(s,1H),6.84(s,1H ),6.78(d,J=8.5Hz,1H),6.73(s,1H),5.13(s,2H),2.77(t,J=6.0Hz,2H) ,2.64(t,J=6.0Hz,2H),1.94-1.90(m,2H),1.28(s,12H).MS(TOF):667.5.

[0028] Example 2:

[0029] Fluorescent probes and ONOO - Solution preparation

[0030] Preparation of probe solution: Weigh a certain amount of probe and dissolve it in dimethyl sulfoxide to prepare a 1×10⁻⁶ solution. -3 M's spare solution. ONOO - Solution preparation: Mix 0.70M H₂O₂ solution, 0.60M HCl solution, and 0.60M NaNO₂ solution, and quickly add 1.5M NaOH solution. Remove excess H₂O₂ with manganese dioxide. Store in a -20°C freezer. Thaw before use. - To determine the concentration, it is necessary to measure the absorbance A of the solution at 302 nm. The calculation formula is: C ONOO -=A / 1.67(mM).

[0031] Example 3:

[0032] Fluorescent probes and ONOO - Determination of the fluorescence spectrum of the effect

[0033] Figure 2 For fluorescent probes and ONOO - The fluorescence spectrum of the action, the concentration of the fluorescent probe is 10 μM, ONOO - The concentrations were 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, and 30 μM, respectively. The excitation wavelength used in the experiment was 650 nm, and the emission wavelength range was 670–900 nm. The slit width was 10.0 nm / 10.0 nm, and the fluorescence measurement instrument used was a Hitachi F4600 fluorescence spectrophotometer. Figure 2 It can be seen that adding ONOO - Previously, due to the quenching effect of borate esters, the probe itself had a low emission peak; with the addition of ONOO... - Subsequently, the emission peak at 725nm is enhanced. Furthermore, with ONOO... - As the concentration increases, the fluorescence intensity of the probe continuously increases. Figure 3 For probes to different ONOO - Linear response graph of concentration. Fluorescence intensity versus ONOO - The concentration showed a linear relationship, with a linear range of 2.5 μM to 30 μM and a detection limit of 0.78 μM. This indicates that the probe can detect ONOO with high sensitivity. - .

[0034] Example 4:

[0035] Fluorescent probes and ONOO - Determination of the UV-Vis absorption spectrum of the effect

[0036] Figure 4 For fluorescent probes and ONOO -The UV-Vis absorption spectrum after treatment; the concentration of the fluorescent probe is 10 μM, ONOO. - The added amount was 30 μM. The UV-Vis absorption spectroscopy measurement was performed using an Agilent Cary 60 UV-Vis spectrophotometer. From... Figure 4 As can be seen, the probe itself has no UV absorption peak; with the addition of ONOO - Subsequently, the probe exhibited an absorption peak at 650 nm.

[0037] Example 5:

[0038] Fluorescent probe for ONOO - Selectivity of the assay

[0039] Figure 5 For fluorescent probes to ONOO - Selectivity curves were obtained. The addition of ONOO to a 10 μM fluorescent probe was investigated. - (30μM) and reactive oxygen species (ClO) - H2O2), various metal ions (Na) + ,K + Ca 2+ Cu 2+ ) and anions (Cl - ,Br - ,I-,NO3 - CO3 2- SO4 2- The fluorescence response of biothiols (Cys, Hcy, GSH) and amino acids (Lys, Phe, Leu, Val, Trp, Lie, Met, Thr) was studied. From... Figure 5 As can be seen from this, only ONOO - The fluorescent probe can induce a significant enhancement of the fluorescence spectrum, while other analytes have no significant effect on the fluorescence spectrum of the probe. These results indicate that the fluorescent probe is effective against ONOO. - It offers good selectivity.

[0040] Example 6:

[0041] Solution pH value affects the determination of fluorescent probe ONOO - Influence of fluorescence properties

[0042] Investigating the effect of pH value on the determination of ONOO by fluorescent probe - The effect of fluorescence spectrum, the results are as follows Figure 6 The pH range we studied was 5.0–10.0, and the concentration of the fluorescent probe was 10 μM. -The concentration was 30 μM. As can be seen from the figure, the fluorescence intensity of the fluorescent probe remained essentially unchanged with pH changes, indicating that pH did not significantly affect the probe itself. However, the addition of ONOO... - Subsequently, the fluorescence intensity ratio significantly increased within the pH range of 7.0–10.0. In conclusion, pH values ​​between 7.0 and 10.0 do not affect the fluorescence probe's response to ONOO. - The determination of the pH value is within a suitable range, which is very beneficial for the application of this probe in actual samples. - The determination.

[0043] Example 7:

[0044] Fluorescent probes and ONOO - Determination of the response time of the action

[0045] Studying fluorescent probes for ONOO - The response time, the result is as follows Figure 7 As can be seen from the figure, the probe is for ONOO. - The response time is 5 minutes, which meets the requirements for real-time monitoring in actual samples. Figure 7 It can also be seen that after the fluorescence intensity reaches its maximum value, the fluorescence intensity does not change again in the following time, which indicates that this fluorescent probe has good photostability.

[0046] Example 8:

[0047] Application of fluorescent probes in living cells

[0048] First, cytotoxicity tests were performed, such as... Figure 8 As shown, when 0–30 μM of the probe was added, the survival rate of HepG2 human liver tumor cells was over 90%. This indicates that the fluorescent probe has low toxicity and can be used to detect ONOO in live cells. - Then, the application of fluorescent probes in living cells was studied. HepG2 human liver tumor cells were selected for confocal microscopy imaging, and the results are as follows: Figure 9 As shown. Adding a fluorescent probe to the cells resulted in almost no fluorescence, indicating that ONOO in the cells... - Lower ( Figure 9 a). Literature reports that lipopolysaccharide (LPS) and interferon-γ (IFN-γ) can jointly stimulate cells to release OONO. - Aminoguanidine hydrochloride (AG) can inhibit intracellular ONOO - The cells were pretreated with LPS and IFN-γ for 10 hours, then stained with a probe for 0.5 hours, resulting in significantly enhanced fluorescence. Figure 9b); Cells were treated with LPS, IFN-γ, and AG for 10 h, then stained with a probe for 0.5 h, resulting in decreased fluorescence ( Figure 9 c). These results demonstrate that the probe can detect ONOO within cells. - Concentration changes. This provides a reliable means of monitoring and treating nitrosamine-related diseases.

Claims

1. A thiazolinone-xanthene peroxynitrite fluorescent probe, i.e. TX-P, based on, characterized in that, The structure is as follows:

2. The preparation method of a thiazolinone-xanthene peroxynitrite fluorescent probe according to claim 1, characterized in that, The reaction steps are as follows: Under the protection of N2, 1 equivalent of TX-OH, 2.0-2.5 equivalents of 4-bromomethylphenylboronic acid pinacol ester, and 2.0-3.0 equivalents of potassium carbonate are sequentially added into a 50 mL round-bottom flask, and then 10-15 mL of DMF is added to dissolve it; stirring at 40-70 DEG C for 5-8 hours; after the reaction is completed, it is extracted with CH2Cl2, the organic phase is collected, dried with anhydrous sodium sulfate, the solvent is removed under reduced pressure, and the obtained crude product is purified by silica gel column chromatography, and the eluent is CH2Cl2 / CH3OH = 80:1-120:1, to obtain black purple solid product TX-P, which is the fluorescent probe; the structure of the above compound TX-OH is as follows:

3. The application according to claim 1, wherein the thiazolinone-xanthene peroxynitrite fluorescent probe is used for the detection of peroxynitrite. The fluorescent probe is used for preparing a detection reagent for peroxynitrite content in living cells.

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