Preparation and application of cyanine dye-based targeted glutathione fluorescent probe

Abstract

The present invention relates to the preparation and application of a targeted glutathione fluorescent probe based on cyanine dyes, and the structural formula of the fluorescent probe is: The present invention provides a preparation method for synthesizing the fluorescent probe using cyanine fluorescent dyes, D-biotin, 4-hydroxy-4-nitroazobenzene, etc. as raw materials; the fluorescent probe is a glutathione fluorescent probe with near-infrared emission, high selectivity and the ability to target cells. First, the fluorescent probe shows high sensitivity to GSH, and the fluorescence is significantly enhanced after the probe reacts with GSH; secondly, the fluorescent probe shows good selectivity to GSH and is not interfered by other common inorganic ions, reactive oxygen, reactive nitrogen, reactive sulfur, amino acids, and biothiols; in addition, the fluorescent probe has been successfully used for GSH imaging in cells and can detect the glutathione level in cells.

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 targeted glutathione fluorescent probe based on cyanine dyes. Background Art

[0002] Glutathione (GSH) is an important biological thiol that plays a key role in regulating various physiological events (SY Zhang, CNOng, HM Shen, Cancer Lett., 2004, 208, 143-153; A. Sharma, MG Lee, M. Won, S. Koo, JF Rambula, JL Sessler, J. Am. Chem. Soc., 2019, 141, 15611-15618). Imbalance of glutathione affects many cell functions, including growth, metabolism, communication and immune response (CT Dooley, TM Dore, GT Hanson, WC Jackson, SJ Remington, RY Tsien, J. Biol. Chem., 2004, 27, 22284-22293). As the most abundant non-protein thiol, glutathione is considered to be a major player in maintaining intracellular redox balance (SUHettiarachchi, B.Prasai, RL McCarley, J.Am.Chem.Soc., 2021, 136, 7575-7578). Therefore, it is of great significance to develop a strategy for efficient detection of GSH.

[0003] So far, many methods for detecting GSH have been developed, such as electrochemical method (YN Zhong, MHLin, JD Zhou, YJ Liu, Chin. J, Anal. Chem., 2010, 38, 229-232), flow injection analysis (A. Waseem, M. Yaqoob, A. Nabi, Chem. Res. Chinese Universities., 2010, 26, 893-898), mass spectrometry (N. Burford, MDE Elman, DE Mahony, M. Morash, Chem. Commun., 2003, 1, 146-147), etc. Compared with these traditional methods, fluorescence analysis method has the advantages of high sensitivity and high resolution imaging of biological samples. So far, many fluorescent probes for detecting GSH have been reported (N. Liu, Y. Jia, MY Chen, CH Guan, Y F Eng, L. Yang, RSC Adv., 2015, 5, 13042-13045; YX Guo, Y. Strongin, R. Mao, Angew. Chem. Ed., 2011, 50, 10690-10693; Z. Xiang, S. Rehm, M. Safont-Sempere, F. Würthner, Nat. Chem., 2009, 1, 623-629; J. Liu, YQ Sun, Y. Huo, H. Zhang, D. Song, W. Guo, J. Am. Chem. Soc., 2014, 136, 574-577). However, these GSH probes have some shortcomings: (1) The analytical wavelength is short, so it is easy to be interfered by the autofluorescence signal generated by biological molecules in vivo, and the tissue penetration ability is weak, which limits the application in vivo; (2) It cannot actively target cells and tissues and is difficult to aggregate in specific tissues. The probes scattered everywhere will reduce the detection efficiency and further enhance the background fluorescence. Therefore, it is very meaningful to design and synthesize GSH fluorescent probes with long wavelength and targeting ability.

[0004] Cyanine dyes are a type of dye that is widely used in the field of fluorescent probes. They have advantages such as large molar absorption coefficient and high photostability. Most importantly, they have near-infrared emission performance. Near-infrared emission can penetrate deeper tissues and is not easily interfered by biological autofluorescence, which is more beneficial for biological imaging. Biotin (vitamin B7) is a well-characterized structure for targeting tumor cells, and various cancer cells, such as cervical cancer, breast cancer, lung cancer, and ovarian cancer, have been reported to overexpress biotin receptors (Y. Singh, K.K. Durga-Rao-Viswanadham, A. Kumar-Jajoriya, J.G. Meher, K. Raval, S. Jaiswal, J. Dewangan, HK Bora, S.K. Rath, J. Lal, Mol. Pharmaceutics, 2017, 14, 2749-2765; K. Li, L. Qiu, Q. Liu, G. Lv, X. Zhao, S. Wang, J. Lin, J. Photochem. Photobiol., 2017, 174, 243-250; N.U. Deshpande, M. Jayakannan, Biomacromolecules, 2018, 19, 3572-3585). Introducing biotin structure into the probe is an effective strategy to achieve tumor-specific distribution. The specific distribution of the probe in cells and tissues is expected to improve the sensitivity and signal-to-noise ratio of GSH detection in cells. However, there is no fluorescent probe that can simultaneously target cancer cells and detect GSH. Therefore, it is very necessary to design and synthesize a cyanine dye-based targeted glutathione fluorescent probe as an effective tool for detecting GSH in cancer cells. Summary of the invention

[0005] According to the requirements put forward, the inventors have conducted in-depth research on this, and after paying a lot of creative work, provided a targeted glutathione fluorescent probe based on cyanine dye.

[0006] The technical solution of the present invention is a targeted glutathione fluorescent probe based on cyanine dye, wherein the structure of Cy-Bio-GSH is as follows:

[0007]

[0008] A method for preparing a targeted glutathione fluorescent probe based on cyanine dye. The steps are as follows:

[0009] 1 equivalent of Cy-Bio-Cl is dissolved in 10-25 mL of anhydrous N,N-dimethylformamide (DMF), added to a 50 mL round-bottom flask, and stirred evenly at -10-0°C. Then, 4-6 equivalents of sodium hydride are slowly added to the round-bottom flask, and 4-6 equivalents of 4-hydroxy-4-nitroazobenzene are dissolved in 4-6 mL of anhydrous DMF, and slowly added dropwise to the above system; then, the reaction system is protected with nitrogen and stirred for 4-6 hours; after the reaction is completed, the crude product is freed from the solvent under reduced pressure, and purified with a CH2Cl2 / CH3OH eluent with a volume ratio of 30:1-10:1 to obtain a blue-green solid compound Cy-Bio-GSH, which is the fluorescent probe.

[0010] The beneficial effect of the present invention is that a targeted glutathione fluorescent probe based on cyanine dye has good spectral response performance. First, the fluorescence spectrum properties of the probe are studied. The fluorescent probe itself has no obvious near-infrared emission peak; after adding GSH, an obvious near-infrared emission peak appears at 820nm. And as the concentration of GSH increases, the near-infrared fluorescence intensity of the probe continues to increase. When the GSH concentration is 40μM, the fluorescence intensity is enhanced by 30 times. The detection range of the probe is from 1μM to 40μM, and the detection limit is 0.3μM, indicating that this fluorescent probe can sensitively detect GSH in the solution. Next, the ultraviolet absorption spectrum of the probe is studied. The probe has no obvious absorption band at 780nm. After adding GSH, the absorption peak appearing near 780nm gradually increases, and the color of the solution changes from light green to blue. Then, the selectivity of the probe is studied. The probe and inorganic ions (Na + ,K + ,Fe 2+ Mg 2+ ), active oxygen (H2O2, ClO - ,O2 - ), active nitrogen (NO2 - ,ONOO - ), active sulfur (H2S, SO3 2- ) and common amino acids (Ala, Lle, Leu, Met, Phe, Pro, Trp, Val, Asn, Gln, Gly, Ser, Thr, Tyr, Arg, Lys) and biological thiols (Cys, Hcy, GSH). The results showed that only GSH could cause changes in the fluorescence spectrum, and other detection substances had no obvious effect on the fluorescence spectrum of the probe. Finally, the effect of pH value on the determination of GSH by the fluorescent probe was studied. As the pH value increased, the fluorescence at 820nm gradually increased. Under the physiological environment of pH = 7.4, the probe had a good response to GSH, indicating that the application of this probe in biological samples has good prospects.

[0011] Application of targeted glutathione fluorescent probe based on cyanine dye. Liver cancer cells that have not been stained with the probe do not emit fluorescence; when the fluorescent probe Cy-Bio-GSH is added to the cells, the fluorescence is significantly enhanced; then, the cells are treated with NEM (intracellular GSH inhibitor) and then stained with the probe Cy-Bio-GSH for imaging, and it is found that almost no fluorescence is generated; the cells are treated with NEM and then with GSH, and finally stained with the probe Cy-Bio-GSH for imaging, which produces strong fluorescence. These results show that the probe Cy-Bio-GSH can sensitively detect intracellular GSH, providing a reliable means for monitoring GSH-related lesions in the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The synthetic route of fluorescent probe.

[0013] Figure 2 The fluorescence spectra of the fluorescent probe after interacting with different concentrations of GSH.

[0014] The horizontal axis is the wavelength, and the vertical axis is the fluorescence intensity. The concentration of the fluorescent probe is 10 μM, and the concentrations of GSH are: 0, 1, 2, 3, 4, 6, 810, 15, 20, 25, 30, 35, 40 μM. The fluorescence excitation wavelength is 780 nm.

[0015] Figure 3 This is the fluorescence linear response diagram of the fluorescent probe to different GSH concentrations.

[0016] Figure 4 This is the UV-visible absorption spectrum of the fluorescent probe before and after the interaction with GSH.

[0017] The concentration of the fluorescent probe was 10 μM, and the concentration of GSH was 40 μM.

[0018] Figure 5 Selectivity diagram of the fluorescent probe.

[0019] The concentration of the fluorescent probe was 10 μM, the concentration of GSH was 40 μM, and the concentration of other analytes was 40 μM.

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

[0021] The concentration of the fluorescent probe was 10 μM, and the concentration of GSH was 40 μM.

[0022] Figure 7 It is the determination of the response time of the fluorescent probe interacting with GSH.

[0023] The concentration of the fluorescent probe was 10 μM, and the concentration of GSH was 0, 10, 20, and 40 μM.

[0024] Figure 8 For cytotoxicity assay.

[0025] The horizontal axis is the concentration of the fluorescent probe, and the vertical axis is the cell survival rate.

[0026] Fig. 9 Cell imaging of the interaction between fluorescent probe and GSH.

[0027] (a) Cells were stained with the probe Cy-Bio-GSH for 30 min; (b) Cells were first treated with NEM for 20 min and then stained with the probe Cy-Bio-GSH for 30 min; (c) Cells were first treated with NEM for 20 min, then treated with 20 μM GSH for 20 min, and then stained with the probe Cy-Bio-GSH for 30 min; (d) Cells were first treated with NEM for 20 min, then treated with 40 μM GSH for 20 min, and then stained with the probe Cy-Bio-GSH for 30 min.

[0028] Fig.10 The relative fluorescence intensity of cells. DETAILED DESCRIPTION

[0029] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but is not limited thereto.

[0030] Embodiment 1:

[0031] Synthesis of fluorescent probes

[0032] Synthesis route such as Figure 1 Synthesis of GSH fluorescent probe (Cy-Bio-GSH): In a 50 mL round-bottom flask, Cy-Bio-Cl (85 mg, 0.1 mmol) was dissolved in 15 mL of anhydrous DMF and stirred evenly at -5 °C; then sodium hydride (13 mg, 0.5 mmol) was slowly added to the round-bottom flask; 4-hydroxy-4-nitroazobenzene (12 mg, 0.5 mmol) was dissolved in 5 mL of anhydrous DMF and slowly added dropwise to the above system; then, the reaction system was protected with nitrogen and continued to stir for 5 h; after the reaction was completed, it was cooled to room temperature, the crude product was freed of solvent under reduced pressure, and purified with CH2Cl2 / CH3OH eluent with a volume ratio of 20:1 to obtain a blue-green solid product (yield 55%), which is the fluorescent probe Cy-Bio-GSH. 1H NMR (400MHz, DMSO-d6) δ8.68(s,1H),8.64(s,1H),8.00(d,J=7.6Hz,2H),7.84(t,J =7.5Hz,2H),7.74(t,J=8.4Hz,4H),7.41(d,J=7.6Hz,2H),6.90-6.82(m,4H),6.68- 6.58(m,6H),6.43(s,1H),6.38(s,1H),3.56-3.42(m,16H),1.40-1.24(m,9H),1.1 8(t,J=6.9Hz,14H),1.09(t,J=7.0Hz,1H),0.88(t,J=6.7Hz,1H).MS(TOF):973.63.

[0033] Embodiment 2:

[0034] Fluorescent probe and GSH solution preparation

[0035] Preparation of probe solution: Weigh a certain amount of probe and dissolve it in dimethyl sulfoxide (DMSO) to make 1×10 -4 Preparation of GSH stock solution: Dissolve a certain amount of GSH in double distilled water to make 1×10 -3 M GSH solution. 1 mL of the probe solution and different volumes of GSH stock solution were added to a 10 mL volumetric flask and fixed to volume with PBS buffer solution at pH = 7.4. -5 M fluorescent probe, 1.0×10 -6 ~4.0×10 -5 M of GSH solution to be tested.

[0036] Embodiment 3:

[0037] Determination of fluorescence spectrum of the interaction between fluorescent probe and GSH

[0038] Figure 2The fluorescence spectrum of the fluorescent probe and GSH is shown in Figure 1. The concentration of the fluorescent probe is 10 μM, and the concentration of GSH is 0, 1, 2, 3, 4, 6, 810, 15, 20, 25, 30, 35, 40 μM. The fluorescence excitation wavelength is 780 nm, and the emission wavelength range is 820-825 nm. The excitation slit width is 5 nm, the emission slit width is 10 nm, and the fluorescence measurement instrument used is a Hitachi F4600 fluorescence spectrophotometer. As can be seen from the figure, due to the quenching effect of the 4-hydroxy-4-nitroazobenzene group, the fluorescent probe itself has no obvious near-infrared emission peak. After adding GSH, an obvious near-infrared emission peak appears at 820 nm. This is because GSH replaces the 4-hydroxy-4-nitroazobenzene of the fluorescent probe, releases the cyanine fluorophore, and thus produces near-infrared fluorescence. Moreover, as the concentration of GSH increases, the near-infrared fluorescence intensity of the probe molecule continues to increase. When the GSH concentration is 40 μM, the fluorescence intensity increases by 30 times. Figure 3 The figure is the linear response graph of the probe to different GSH concentrations. The fluorescence intensity is linearly related to the concentration of GSH, the linear range is 1-40 μM, and the detection limit is 0.3 μM. This shows that the probe can detect GSH with high sensitivity.

[0039] Embodiment 4:

[0040] Determination of UV-visible absorption spectrum of fluorescent probe interacting with GSH

[0041] Figure 4 The figure is the UV-visible absorption spectrum after the fluorescent probe and GSH react. The concentration of the fluorescent probe is 10μM and the amount of GSH added is 40μM. The instrument used for UV-visible absorption spectrum measurement is Agilent Cary60 UV-visible spectrophotometer. It can be seen from the figure that the probe has no obvious absorption band at 780nm. After adding GSH, the absorption peak near 780nm gradually increases.

[0042] Embodiment 5:

[0043] Selectivity of fluorescent probes for GSH determination

[0044] Figure 5 The selectivity of the fluorescent probe for GSH was investigated by adding GSH (40 μM) and inorganic ions (Na + ,K + ,Fe 2+ Mg 2+ ), active oxygen (H2O2, ClO - ,O2 - ), active nitrogen (NO2 - ,ONOO - ), active sulfur (H2S, SO32- ) and common amino acids (Ala, Lle, Leu, Met, Phe, Pro, Trp, Val, Asn, Gln, Gly, Ser, Thr, Tyr, Arg, Lys) and biothiols (Cys, Hcy, GSH). As can be seen from the figure, only GSH 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 good selectivity for GSH.

[0045] Embodiment 6:

[0046] Effect of Solution pH on the Fluorescence Properties of GSH Determined by Fluorescence Probe

[0047] The effect of pH on the fluorescence spectrum of GSH measured by fluorescent probe was investigated. Figure 6 . The pH range we studied was 4.0-11.0, the concentration of the fluorescent probe was 10 μM, and the concentration of GSH was 40 μM. As can be seen from the figure, the fluorescence of the fluorescent probe is always in the off state as the pH increases, indicating that pH has no great effect on the probe itself. However, after the addition of GSH, the fluorescence intensity significantly increased with the increase in pH, and gradually decreased when the pH reached 8 and above. However, within the physiological pH range of 7.0-8.0, the fluorescence intensity is basically stable. In summary, this probe has the potential for testing biological samples.

[0048] Embodiment 7:

[0049] Determination of the response time of the fluorescent probe interacting with GSH

[0050] We studied the response time of the fluorescent probe to GSH, and the results are as follows Figure 7 As can be seen from the figure, the response time of the probe to GSH is 15 minutes, which can meet the requirements of real-time monitoring in actual samples. Figure 7 It can also be seen that after the fluorescence intensity reaches the maximum value, the fluorescence intensity no longer changes in the following period of time, which indicates that this fluorescent probe has good photostability.

[0051] Embodiment 8:

[0052] Application of fluorescent probes in living cells

[0053] First, we performed cytotoxicity assays, e.g. Figure 8 As shown. When 0-30 μM fluorescent probe was added, the cell survival rate was above 90%. This shows that the fluorescent probe has low toxicity and can be used to detect GSH in living cells. Then, the application of fluorescent probe in living cells was studied, and liver cancer cells were selected for confocal microscopy imaging. The results are shown in Fig. 9As shown. After cells were stained with the probe Cy-Bio-GSH for 30 minutes, the fluorescence was significantly enhanced ( Fig. 9 a); Subsequently, the cells were treated with NEM for 20 min and then stained with the probe Cy-Bio-GSH for 30 min for imaging, and it was found that almost no fluorescence was generated ( Fig. 9 b); The cells were treated with NEM for 20 min and then with 20 μM GSH for 20 min. The probe Cy-Bio-GSH was used to stain and image for 30 min, which produced fluorescence ( Fig. 9 c); treated with 40 μM GSH for 20 min, and finally stained with probe Cy-Bio-GSH for 30 min, which produced strong fluorescence ( Fig. 9 d). Fig.10 These results show that the probe Cy-Bio-GSH can sensitively detect intracellular GSH, which provides a reliable means for monitoring the GSH content in biological samples.

Claims

1. A targeted glutathione fluorescent probe based on cyanine dye, wherein: The structure of Cy-Bio-GSH is as follows:

2. The method for preparing a cyanine dye-based targeted glutathione fluorescent probe according to claim 1, characterized in that: The reaction steps are as follows: Dissolve 1 equivalent of Cy-Bio-Cl in 10-25 mL of anhydrous N,N-dimethylformamide (DMF), add to a 50 mL round-bottom flask, and stir evenly at -10-0°C. Then slowly add 4-6 equivalents of sodium hydride to the round-bottom flask, dissolve 4-6 equivalents of 4-hydroxy-4-nitroazobenzene in 4-6 mL of anhydrous DMF, and slowly dropwise add to the above system; then, protect the reaction system with nitrogen and continue stirring for 5 hours; after the reaction is completed, remove the solvent from the crude product under reduced pressure, and purify it with a CH2Cl2 / CH3OH eluent with a volume ratio of 30:1-10:1 to obtain a blue-green solid compound Cy-Bio-GSH, which is the fluorescent probe, wherein the structure of Cy-Bio-Cl is as follows:

3. The use of a cyanine dye-based targeted glutathione fluorescent probe according to claim 1, characterized in that: The fluorescent probe is used for detecting the intracellular glutathione content for purposes other than disease diagnosis and treatment.

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