Synthesis and application of coumarin-xanthene-based polar fluorescent probe

Abstract

The invention relates to synthesis and application of a fluorescent probe based on coumarin-xanthene. The structural formula of the probe is shown in the specification. The invention provides a preparation method for synthesizing the fluorescent probe by taking coumarin and xanthene as raw materials. The fluorescent probe is a polar fluorescent probe with high sensitivity, high selectivity and low cytotoxicity; firstly, the fluorescent probe has good sensitivity to fluorescence response of system polarity change; secondly, the fluorescent probe is not interfered by various other ions, active oxygen, biological mercaptan and amino acid, and only shows good selectivity to polarity; moreover, the fluorescent probe has better light stability, and the fluorescence intensity is not changed under continuous illumination for 120 minutes; when the pH value is 7.4, the fluorescence signal of the probe is the strongest, and the change of polarity can be detected in a complex physiological environment; in addition, the fluorescent probe is low in cytotoxicity and can be applied to detection of polarity change in living cells.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to the synthesis and application of a polar fluorescent probe based on coumarin-oxanthracene. Background Technology

[0002] Polarity, as an important parameter of the cellular microenvironment, participates in various physiological processes, including protein denaturation, enzyme catalysis, peptide aggregation, membrane fusion, and signal transduction (Fan L, Wang X, Zan Q, et al. Lipid droplet-specific fluorescent probe for in vivo visualization of polarity in fatty liver, inflammation, and cancer models[J]. Analytical Chemistry, 2021, 93: 8019-8026). Furthermore, abnormalities in polarity are closely associated with various diseases, such as Alzheimer's disease, diabetes, cirrhosis, and cancer (Perez RG, Zheng H, Van der Ploeg LHT, et al. The β-Amyloid precursor protein of alzheimer's disease enhances neuron viability and modulates neuronal polarity[J]. The Journal of Neuroscience, 1997, 17: 9407). Therefore, accurate detection of polarity levels is crucial in the cellular and even biomedical fields (Miao J, Huo Y, Yao G, et al. Heavy atom-free, nitochondria-targeted, and activatable photosensitizers for photodynamic therapy with real-time in-situ therapeutic monitoring[J]. Angewandte Chemie International Edition, 2022, 61: 2022-01815).

[0003] Ideal tools for polarity detection remain scarce. Small molecule fluorescent probes, with their high sensitivity and other properties, have made remarkable progress in the past decade, becoming the main research tool for detecting polarity in the cellular microenvironment and medical fields (Ma Q, Zhang Y, Jiao Y, et al. New β-diketone-boron difluoride based near-infraredfluorescent probes for polarity detection[J]. Analyst, 2021, 146: 5873-5879; Hu GD, Jia HY, Zhao LN, et al. Small molecule fluorescent probes of protein vicinal dithiols[J]. Chinese Chemical Letters, 2019, 30: 1704-1716). Currently, several fluorescent probes for monitoring polarity have been reported (Hu L, Shi D, Li X, et al. Curcumin-based polarity fluorescent probes: design strategy and biological applications[J]. Dyes and Pigments, 2020, 177: 108320; Xiao H, Li P, Tang B. Recent progresses in fluorescent probes for detection of polarity[J]. Coordination Chemistry Reviews, 2021, 56: 427; Lu B, Yin J, Liu C, et al. Lipid droplet polarity decreases during the pathology of muscle injury as revealed by a polarity sensitive sensor[J]. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2021, 26: 2120149).However, these polar fluorescent probes still have some limitations. Most rely on short-chain conjugated structures, resulting in short emission wavelengths and shallow imaging depths, thus limiting their applications (Li X, Li X, Ma H, et al. Anear-infrared fluorescent probe reveals decreased mitochondrial polarity during mitophagy[J]. Chemical Science, 2020, 11: 1617-1622). Ideal polarity detection tools are yet to be developed to monitor polarity in biological microenvironments.

[0004] Coumarin-oxanthracene, as a novel fluorescent dye, possesses a long emission wavelength, thus exhibiting deeper tissue penetration and being less susceptible to interference from biological autofluorescence, making it more advantageous for bioimaging. However, until now, no fluorescent probe based on coumarin-oxanthracene dyes has been developed for detecting polarity. Therefore, this invention designs and synthesizes a fluorescent probe based on coumarin-oxanthracene for detecting polarity. 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 polar fluorescent probe based on coumarin-oxanthracene.

[0006] The technical solution of this invention is a polar fluorescent probe based on coumarin-oxanthracene, the structural formula of which is as follows:

[0007]

[0008] A method for preparing a polar fluorescent probe based on coumarin-oxanthracene. The steps are as follows:

[0009] In a 50 mL round-bottom flask, 1.0 equivalent of xanthene and 1.0–1.5 equivalents of coumarin were dissolved in toluene. Under nitrogen protection, 0.2–0.4 mL of piperidine was added, and the reaction was carried out at 70–80 °C for 12–18 h. After the reaction was completed, the mixture was cooled to room temperature, and the solvent was removed by vacuum distillation. The crude product was purified by column chromatography using dichloromethane:methanol = 100:1–50:1 as the eluent to obtain the red solid product CX-P, which is the fluorescent probe.

[0010] The beneficial effect of this invention is the excellent spectral response performance of a coumarin-oxanthracene-based polar fluorescent probe. Solutions of different polarities were obtained by adjusting the ratio of water and 1,4-dioxane, and evaluated using the Lippert-Matagassal polarity parameter Δf. Fluorescence spectra under different solvent polarities showed that as the solvent polarity increased from Δf = 0.088 (99% 1,4-Dionxane) to Δf = 0.321 (0% 1,4-Dionxane), the maximum emission wavelength of CX-P redshifted from 625 nm to 694 nm, and the fluorescence intensity gradually decreased. Simultaneously, when the polarity Δf of the test system varied within the range of 0.088 to 0.321, the fluorescence intensity (F) at the maximum emission wavelength position... max The probe exhibited a good linear relationship with polarity (Δf), indicating that it is highly sensitive to polarity. Next, the UV absorption spectrum of the probe was investigated. The maximum absorbance of the probe in H₂O was at 475 nm, while the maximum absorbance wavelength in 1,4-Dionxane red-shifted to 500 nm. Subsequently, the selectivity of the probe was studied, examining various analytes, such as reactive oxygen species (H₂O₂, ClO₂). - ), reactive nitrogen (NO - NO2 - ONOO - ), active sulfur (HS) - HSO3 - ), anion (I - ,Br - ,Cl - CH3COO - SO4 2- CO3 2- ), cations (K) + Mg 2+ Na + ,Fe 2+ Ca 2+ Cu 2+ ,Zn 2+ NH4 + The response of probe CX-P in the presence of biothiols (GSH, Cys, Hcy) and amino acids (Val, Ile, Met, Thr, Trp, Phe, Leu, Lys, His) was investigated. The study found that probe CX-P exhibited good selectivity for polarity compared to the aforementioned interfering substances. The effect of different pH values ​​on the polarity determination of CX-P was then explored in detail. The probe showed the strongest fluorescence signal at pH 7.4, indicating that CX-P can detect changes in polarity under complex physiological conditions. Furthermore, this fluorescent probe possesses excellent photostability, ensuring its practicality.

[0011] Application of a coumarin-oxanthracene-based polar fluorescent probe. Experiments were conducted using 4T1 cells. The control group, cultured with only 10 μM CX-P, showed almost no fluorescence. Pretreatment of cells with lipopolysaccharide (LPS) or oleic acid (OA), followed by incubation with the probe, resulted in a significant increase in fluorescence in cells induced by LPS or OA, indicating that the probe responds to polarity in living cells. Attached Figure Description

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

[0013] Figure 2 The images show the fluorescence spectra of the fluorescent probe in solutions of different polarities.

[0014] The x-axis represents wavelength, and the y-axis represents fluorescence intensity. The concentration of the fluorescent probe was 10 μM, and the percentages of 1,4-Dioxane in water were 0%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 99%, respectively. The corresponding Δf values ​​were 0.321, 0.303, 0.291, 0.264, 0.245, 0.222, 0.200, 0.182, 0.159, 0.137, 0.114, and 0.088, respectively. The emission wavelength ranged from 625 to 694 nm, and the corresponding excitation wavelength was 500 nm.

[0015] Figure 3 This is a graph showing the relationship between the logarithm of the fluorescence intensity of the fluorescent probe and the logarithm of Δf.

[0016] The x-axis represents Δf, and the y-axis represents fluorescence intensity. The concentration of the fluorescent probe is 10 μM.

[0017] Figure 4 This shows the UV-Vis absorption spectra of the fluorescent probe in solutions of different polarities.

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

[0019] The concentration of the fluorescent probe was 10 μM, and the concentration of other analytes was 200 μM.

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

[0021] Figure 7 This is a photostability graph of the fluorescent probe.

[0022] Figure 8 This 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 9Cellular imaging of the fluorescent probe.

[0024] Figure 10 Relative fluorescence intensity diagram. Detailed Implementation

[0025] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but is not limited thereto.

[0026] Example 1:

[0027] Synthesis of fluorescent probes

[0028] Synthetic routes such as Figure 1 In a 50 mL round-bottom flask, 1.0 equivalent of xanthene and 1.0 equivalent of coumarin were dissolved in toluene. Under nitrogen protection and stirring, 0.2 mL of piperidine was added. The reaction was carried out at 80 °C for 16 h. After the reaction was completed, the mixture was cooled to room temperature, and the solvent was removed by vacuum distillation. The crude product was purified by column chromatography using dichloromethane:methanol = 50:1 as the eluent to obtain a red solid product CX-P, which is the fluorescent probe (30 mg, yield 16%). 1 H NMR (400MHz, CDCl3) δ 8.52 (s,

[0029] 1H),8.05(d,J=15.1Hz,1H),7.41(d,J=8.9Hz,1H),7.32(d,J=15.2Hz,1H),6.93(d,J=8.4Hz,1H),

[0030] 6.68–6.53(m,3H),6.47(d,J=2.4Hz,1H),6.32(s,1H),3.82(s,3H),3.45(q,J=7.1Hz,4H),2.79–2.69(m,4H),1.24(d,J=7.0Hz,6H). 13 C NMR (101MHz, CDCl3) δ185.76,160.95,160.30,158.36,157.88,153.61,152.54,147.9 0,138.40,134.96,131.48,126.68,120.92,118.11,117.79,116.28,116.00,110.16,

[0031] 109.62,108.77,101.44,96.61,55.58,45.11,26.02,24.55,12.51.MS(TOF):470.5.

[0032] Example 2:

[0033] Preparation of fluorescent probes and solutions of 1,4-dioxane in different proportions and calculation of Δf

[0034] Preparation of probe solution: Weigh a certain amount of probe and dissolve it in dimethyl sulfoxide to prepare a 1×10⁻⁶ solution. -4 A spare solution of mol / L was prepared. 1.0 mL of the probe's spare solution was added to a 10 mL volumetric flask, and the volume was adjusted to 1.0 × 10⁻⁶ mol / L with buffer solution to obtain a concentration of 1.0 × 10⁻⁶ mol / L. -5 Fluorescent probe solutions of mol / L were prepared. Solutions with different percentages of 1,4-Dioxane in water (0%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%) were prepared, with corresponding Δf values ​​of 0.321, 0.303, 0.291, 0.264, 0.245, 0.222, 0.200, 0.182, 0.159, 0.137, 0.114, and 0.088, respectively.

[0035] Example 3:

[0036] Determination of fluorescence spectra of fluorescent probes in solutions of different polarities

[0037] Figure 2 The fluorescence spectra of the fluorescent probe in solutions of different polarities are shown. The concentration of the fluorescent probe was 10 μM, and the percentages of 1,4-Dioxane in the solutions were 0%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 99%, respectively. The excitation wavelength used in the experiment was 500 nm, and the emission wavelength range was 625–694 nm. The fluorescence measurement instrument used was an F-4600 fluorescence spectrometer. As can be seen from the figure, when the probe is in aqueous solution, there is almost no emission peak; as the percentage of 1,4-Dioxane increases, that is, as the polarity of the solution decreases, the fluorescence gradually increases. Figure 3 This is a linear response graph of the probe to different polarities. Fluorescence intensity and Δf show a linear relationship, ranging from 0.088 (99% 1,4-Dionxane) to 0.321 (0% 1,4-Dionxane). This probe has a wide detection range and can meet the detection requirements for different polarities.

[0038] Example 4:

[0039] Determination of UV-Vis absorption spectra of fluorescent probes in solutions of different polarities

[0040] Figure 4The images show the UV-Vis absorption spectra of a fluorescent probe at different polarities. The probe concentration was 10 μM, and the solutions were water and 1,4-Dioxane. The UV-Vis absorption spectra were measured using an Agilent Cary 60 UV-Vis spectrophotometer. Figure 4 As can be seen, the maximum absorbance of the probe in H2O is at 475 nm, while the maximum absorbance wavelength in 1,4-Dioxane is red-shifted to 500 nm.

[0041] Example 5:

[0042] Selectivity of fluorescent probes for polarity determination

[0043] Figure 5 To ensure the selectivity of the fluorescent probe for polarity, the probe interacts with various analytes, such as reactive oxygen species (H2O2, ClO2). - ), reactive nitrogen (NO - NO2 - ONOO - ), active sulfur (HS) - HSO3 - ), anion (I - ,Br - ,Cl - CH3COO - SO4 2- CO3 2- ), cations (K) + Mg 2+ Na + ,Fe 2+ Ca 2+ Cu 2+ ,Zn 2+ NH4 + The response of probe CX-P in the presence of biothiols (GSH, Cys, Hcy) and amino acids (Val, Ile, Met, Thr, Trp, Phe, Leu, Lys, His) was investigated. The results showed that only 1,4-Dioxane caused a change in the fluorescence spectrum; other interfering substances had no significant effect on the probe's fluorescence spectrum, indicating that this fluorescent probe exhibits good polarity selectivity.

[0044] Example 6:

[0045] The effect of solution pH on the fluorescence properties of fluorescent probes that determine polarity

[0046] The effect of pH on the fluorescence spectrum of a fluorescent probe determining polarity was investigated, and the results are as follows: Figure 6Our study covered a pH range of 4–10, with a fluorescent probe concentration of 10 μM. As shown in the figure, in aqueous solution, the probe fluorescence intensity remained essentially constant with pH changes. When the probe was added to a 50% 1,4-Dioxane solution, the solution polarity decreased. As the solution alkalinity increased, the fluorescence weakened. These experimental results demonstrate that within the physiological pH range, the probe maintains a good response to polarity and does not affect the determination of polarity in the fluorescent probe. This is highly advantageous for using this probe to determine the polarity of biological samples.

[0047] Example 7:

[0048] Determination of photostability of fluorescent probes in 1,4-Dionxane

[0049] We investigated the photostability of the fluorescent probe in 1,4-Dionxane solution. Measurements were taken from the moment the probe was added to the solution until 2 hours had elapsed. The results are as follows: Figure 7 As can be seen from the figure, the probe maintains a good polarity response and exhibits good photostability even after 2 hours in 1,4-Dionxane, which meets the requirements for detection in actual samples.

[0050] Example 8:

[0051] Application of fluorescent probes in living cells

[0052] First, we conducted cytotoxicity tests, such as... Figure 8 As shown, when 0–30 μM probe was added, the survival rate of 4T1 cells was over 90%. This indicates that the fluorescent probe has low toxicity and can be used to detect polarity in live cells. Then, to study the imaging ability of CX-P on changes in cell polarity, cell polarity was induced by LPS or OA, respectively, and the results were obtained from… Figure 9 As can be seen, the fluorescence signal in the control group cell images is very weak; in contrast, clear and bright red fluorescence can be seen in the LPS or OA group cell images. Figure 10 The relative fluorescence intensities of the three groups of cells were shown, demonstrating that the probe can detect intracellular polarity with high sensitivity.

Claims

1. A polar fluorescent probe based on coumarin-xanthene, i.e. CX-P, characterized in that, The structure is as follows:

2. A synthesis of a polar fluorescent probe based on coumarin-xanthene according to claim 1, characterized in that, The reaction steps are as follows: In a 50 mL round-bottom flask, 1.0 equivalent of xanthene and 1.0-1.5 equivalents of xanthene are dissolved in toluene, 0.2-0.4 mL of piperidine is added under nitrogen protection, 70-80 ℃, reaction for 12-18 h, after the reaction is completed, it is cooled to room temperature, the solvent is removed by reduced pressure distillation, and the obtained crude product is purified by column chromatography, with dichloromethane:methanol = 100:1-50:1 as an eluent, to obtain a red solid product CX-P, which is the fluorescent probe.

3. The use of a polar fluorescent probe based on coumarin-xanthene according to claim 1, characterized in that, The fluorescent probe can be applied to the detection of polarity in living cells.

Images