A dual-channel near-infrared ratio fluorescent probe for detecting tyrosinase and a preparation method and use thereof

Abstract

The application discloses a kind of detection tyrosinase dual-channel near-infrared ratio fluorescent probe and its preparation method and purposes, wherein the structure of detection tyrosinase dual-channel near-infrared ratio fluorescent probe is as follows: the dual-channel near-infrared ratio fluorescent probe LZD of the application shows good selectivity and sensitive dual-channel fluorescence signal to tyrosinase.Cytotoxicity test shows that the ratio probe has good biocompatibility, and confocal fluorescence microscopic imaging experiment shows that the ratio probe has good light stability to HepG2 cells, can respond to tyrosinase through dual-channel fluorescence, and can carry out confocal fluorescence imaging to extracellular and endogenous tyrosinase of cell.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probes, specifically relating to a dual-channel near-infrared ratiometric fluorescent probe for detecting tyrosinase, its preparation method and application, to achieve dual-channel near-infrared fluorescence imaging of tyrosinase in cells, with the advantages of selectivity, high detection efficiency and good biocompatibility. Background Technology

[0002] As the basic unit of life, the cell is an ingenious and complex chemical machine that performs various biological activities. Various small and large biological molecules within the cell coordinate with each other to perform a variety of complex biological functions. Enzymes are one of the most important classes of key macromolecules within the cell, playing a crucial role in various catalytic reactions essential for normal life. Tyrosinase, a ubiquitous copper-containing oxidase, is widely found in microorganisms, plants, animals, and other biological systems. It catalyzes the hydroxylation of tyrosine or tyrosine, further oxidizing the substrate to yield the corresponding ortho-quinone products in living systems. Numerous studies have shown that imbalances in tyrosinase levels can lead to many serious diseases. Furthermore, upregulated tyrosinase is considered an important biomarker for malignant melanoma and liver cancer and has been extensively studied. Therefore, detecting tyrosinase activity is of great significance.

[0003] Currently, many fluorescent probes for tyrosinase (TYR) detection have been developed due to their simple preparation, high sensitivity, high selectivity, and live-cell imaging capabilities. However, most of these probes are designed for the diagnosis of malignant melanoma and have significant drawbacks, such as small Stokes shift and single-channel imaging, which may result in strong autofluorescence and poor fluorescence signal-to-noise ratio. These limitations can severely affect the detection and imaging of tyrosinase in vitro and in vivo. Therefore, there is an urgent need for a novel tyrosinase ratio fluorescent probe that can successfully overcome these drawbacks and distinguish tyrosinase levels in different cell lines. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a dual-channel near-infrared ratiometric fluorescent probe for detecting tyrosinase, its preparation method, and its applications. The technical problem to be solved is to obtain a ratiometric probe that can perform dual-channel fluorescence imaging and identify the activity of exogenous and endogenous tyrosinases through molecular design. It has the advantages of selectivity and specificity, good photostability, dual-channel imaging, and near-infrared imaging. Cytotoxicity tests show that the ratiometric probe of this invention has good cell compatibility.

[0005] This invention presents a dual-channel near-infrared ratiometric fluorescent probe that determines intracellular tyrosinase levels by detecting changes in tyrosinase activity. Furthermore, the electron-accepting quinoline cation is linked to an xanthracene derivative, forming a donor-acceptor (D-π-A) structure that favors the transition from red fluorescence to the near-infrared emission region and a large Stokes shift. Based on this design strategy, we successfully synthesized probe LZD, an NIR ratiometric fluorescent probe. Upon response to tyrosinase in living cells, probe LZD partially transforms from a red fluorophore to a green fluorophore, thus exhibiting excellent dual-channel imaging capabilities. It also demonstrates high sensitivity and selectivity for TYR. This ratiometric probe has been applied to measure TYR activity in cells with significant results.

[0006] This invention relates to a dual-channel near-infrared ratiometric fluorescent probe, abbreviated as LZD, which uses an oxanthracene derivative as its parent material, and its structural formula is shown below:

[0007]

[0008] The preparation method of the dual-channel near-infrared ratiometric fluorescent probe of the present invention includes the following steps:

[0009] Step 1: Mix 30 mL of N,N-dimethylformamide (DMF) with 10 mL of dichloromethane (DCM), add phosphorus tribromide dropwise at 0 °C, remove from ice-water bath and add cyclohexanone dropwise; after reacting for six hours, add the reaction solution dropwise to 150 mL of water, then neutralize with sodium carbonate, extract with water and DCM, and evaporate to dryness to obtain intermediate 1;

[0010] Step 2: Add 9.4g of intermediate 1, 5.3g of 2-hydroxy-4-methoxybenzaldehyde, and 17g of potassium carbonate to 30ml of DMF and react for 12h. Then extract with water and DCM, evaporate to dryness, and slurry (ethyl acetate: petroleum ether = 1:1, v / v). Filter to obtain intermediate 2.

[0011] Step 3: Add 0.5 g of 3-hydroxybenzyl alcohol to 15 mL of DCM, add phosphorus tribromide dropwise at 0 °C, react at room temperature for 2 h, dilute with 25 mL of DCM, extract (water / DCM), and rotary evaporate to obtain intermediate 3;

[0012] Step 4: Add 2.25 g of intermediate 3 and 1.7 g of tetramethylquinoline to 25 mL of ethanol, reflux at 80 °C for 12 h, evaporate the solvent by rotary evaporation, and separate by column chromatography (methanol: dichloromethane = 1:20, v / v) to obtain intermediate 4;

[0013] Step 5: Add 200 mg of intermediate 2, 206 mg of intermediate 4 and two drops of piperidine to 5 mL of ethanol, reflux at 80 °C for 12 h, evaporate the solvent by rotary evaporation, and slurry with ethyl acetate to obtain intermediate 5;

[0014] Step 6: Add 100 mg of intermediate 5 and 42 mg of TEA to 2 ml of DCM, add 35 mg of phosphorus tribromide dropwise at 0 °C, react overnight at room temperature, evaporate the solvent, and separate by column chromatography (methanol: dichloromethane = 1:20, v / v) to obtain the target product LZD.

[0015] The synthetic route of this invention is shown below:

[0016]

[0017] The purpose of the dual-channel near-infrared ratiometric fluorescent probe of the present invention is for the preparation of tyrosinase detection reagents for non-therapeutic or diagnostic purposes. The detection reagents can detect endogenous and exogenous tyrosinases in cells using dual-channel imaging.

[0018] Furthermore, when detecting tyrosinase with the aforementioned detection reagent, the ratio of fluorescence intensity at 715 nm to fluorescence intensity at 510 nm in the detection system (I 715 / I 510 There is a linear relationship between the concentration of tyrosinase and the concentration of tyrosinase.

[0019] The detection method is as follows:

[0020] The LZD of this invention was dissolved in DMSO to prepare a 2 mM stock solution. 15 μL of this stock solution was then added to 3 mL of Hepes solvent containing different concentrations of tyrosinase. Fluorescence and UV spectra of 10 μL LZD in different test solutions were obtained. As the tyrosinase concentration decreased, the absorbance of LZD increased at 420 nm and decreased at 600 nm. Using 420 nm and 600 nm as excitation wavelengths, the fluorescence decreased at 510 nm and increased at 715 nm, indicating its dual-channel imaging capability. Molecular docking simulations of LZD were then performed, showing that it can bind firmly to tyrosinase through hydrogen bonds and intermolecular forces. Dark cytotoxicity experiments were conducted using the MTT (5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) method. LZD was added to live HepG2 cells at various concentrations (0 μM, 5.0 μM, 10.0 μM, 15.0 μM, 20.0 μM) and incubated in the dark for 24 hours. Cell viability was not affected. To investigate the imaging capability of LZD for tyrosinase in cells, cells with added LZD were monitored at different time points. No changes were observed in the fluorescence of the red and green channels. These results indicate that LZD can stably image cells using a dual-channel approach. Further investigation into the dual-channel detection capability of LZD for tyrosinase in cells revealed that, compared to samples containing tyrosinase, the control group showed decreased green emission and increased red emission. Subsequently, the addition of a tyrosinase inhibitor to the samples resulted in decreased green emission and increased red emission in the control group. These results demonstrate that LZD can perform dual-channel endogenous detection of tyrosinase within cells.

[0021] This invention utilizes near-infrared dual-channel fluorescence detection to achieve fluorescence imaging of tyrosinases in cells. It exhibits stable fluorescence imaging capabilities, and cytotoxicity tests demonstrate good cell compatibility with LZD. Confocal fluorescence microscopy experiments show that LZD can perform dual-channel fluorescence imaging of tyrosinases. Attached Figure Description

[0022] Figure 1 The response of LZD (10 μM) to TYR is shown. Among them: (a) absorption spectrum of LZD (10 μM); (b) fluorescence spectrum of LZD (10 μM) at 510 nm; (c) fluorescence spectrum of LZD (10 μM) at 715 nm; (d) linear relationship between fluorescence intensity of LZD (10 μM) and different TYRs.

[0023] Figure 2 This is the probe time response of LZD.

[0024] Figure 3 This refers to the probe selectivity of LZD.

[0025] Figure 4 This refers to the pH stability of the LZD probe.

[0026] Figure 5 This is a simulation test of molecular docking between LZD and tyrosinase. (a) Molecular docking of LZD with TYR; (b) Enlarged view of the region in (a).

[0027] Figure 6 The image shows the survival rate of HepG2 cells under different concentrations (0 μM, 5 μM, 10 μM, 15 μM, 20 μM) of LZD.

[0028] Figure 7 These are confocal fluorescence images of 10 μM LZD, used to investigate the optical stability of LZD. (a) Fluorescence images of HepG2 cells stained with LZD (10 μM) at different time points. (b) Fluorescence intensities of the blue and red channels in (a).

[0029] Figure 8 These are confocal fluorescence images of 10 μM LZD, used to investigate the exogenous response of LZD to TYR. (a) Fluorescence image of HepG2 cells co-stained with LZD (10 μM) and tyrosinase. (b) Fluorescence intensities of the blue and red channels in (a).

[0030] Figure 9These are confocal fluorescence images of 10 μM LZD to investigate the endogenous response of LZD to TYR. (a) Fluorescence image of HepG2 cells stained with LZD (10 μM) and kojic acid. (b) Fluorescence intensities of the blue and red channels in (a). Detailed Implementation

[0031] The present invention will be further illustrated by the following examples.

[0032] Example 1: Synthesis of LZD

[0033] 100 mg of intermediate 5 and 42 mg of TEA were added to 2 ml of DCM, and 35 mg of phosphorus tribromide was added dropwise at 0 °C. The reaction was carried out overnight at room temperature, the solvent was evaporated, and the product was obtained by column chromatography (methanol: dichloromethane = 1:20) to give the target product LZD.

[0034] 1 H NMR (400MHz, DMSO-d, ppm) δ9.18(d,J=6.8Hz,1H),8.78(d,J=8.7Hz,1H),8.54(d,J=15.0Hz,1H),8.41(d,J=6.8 Hz,1H),8.20(d,J=8.9Hz,1H),8.04(d,J=7.9Hz,2H),7.88(d,J=7.6Hz,1H),7.79(t,J=7.8Hz,1H),7.69(s,1H), 7.54(s,2H),7.46(s,1H),7.39(s,1H),7.29(d,J=6.6Hz,2H),7.24(s,2H),7.07(d,J=2.4Hz,1H),6.89(s,1H),6 .73(dd,J=8.5,2.5Hz,1H),6.16(s,2H),3.80(s,3H),2.66(t,J=6.1Hz,2H),2.55(t,J=6.1Hz,2H),1.73(s,2H). 13C NMR(151MHz,DMSO-d,ppm)δ164.97,161.41,155.35,154.20,153.53,151.57,146.74 ,138.83,138.49,137.17,135.10,134.72,130.85,130.32,129.53,129.18,128.87, 128.29,128.02,127.66,127.55,126.90,126.57,125.16,122.71,121.06,119.52,115.54,114.67,114.43,113.30,111.84,101.27,58.25,56.35,29.27,24.73,20.81.

[0035] Example 2: LZD response to tyrosinase

[0036] Since fluorescence intensity depends on the absorption level of the ratiometric probe, we tested the changes in UV-Vis absorption spectra of TZD in response to a series of TYR signals. With increasing TYR concentration, the UV-Vis absorption of LZD at 620 nm gradually decreased, indicating that the fluorescence intensity at 715 nm gradually decreased when 620 nm was used as the excitation wavelength. Conversely, the absorption in the visible region (420 nm) gradually increased, confirming that the fluorescence intensity at 510 nm gradually increased when 480 nm was used as the excitation wavelength. Figure 1 (a)

[0037] like Figure 1 As shown in Figures b and c, as expected, the 715 nm emission peak gradually decreases with increasing TYR concentration (λ). ex =620nm), while the emission peak is at 510nm (λ). ex The wavelength (λ = 420 nm) gradually increases. This dual-excitation-dual-emission system avoids the experimental biases widely present in single-excitation-dual-emission systems. Based on this, we have constructed a precise detection method for TYR using a dual-excitation-dual-emission ratiometric fluorescence strategy, with a detection limit as low as 0.42 U / mL. Figure 1 The middle d shows the ratio of fluorescence intensity at 510 nm to fluorescence intensity at 715 nm (I 715 / I 510 The correlation coefficient shows a good linear relationship and is R. 2 =0.991.

[0038] Example 3: Probe Time Response of LZD

[0039] The results of testing the LZD probe at different TYR concentrations over time showed that fluorescence stabilized after 1.5 min, which is beneficial for the sensitive detection of tyrosinase. Figure 2 ).

[0040] Example 4: Probe selectivity of LZD

[0041] LZD probes are used to measure various analytes, including Mg. 2+ Na + Ga 2+ Cl - SO4 2- HSO3 2- , H2S, Gly, Cys, Hcy, GSH, ClO - H2O2, BSA, GGT, TYR. Analytical results ( Figure 3 The results indicate that LZD has a significant ratiometric fluorescence attenuation effect on tyrosinase, while the fluorescence changes of other analytes are negligible. These results demonstrate that LZD exhibits extremely high selectivity for tyrosinase.

[0042] Example 5: Probe pH stability of LZD

[0043] To address the potential interference of the LZD probe's fluorescence in a weakly alkaline environment after targeting mitochondria, the pH stability of the probe was tested. Figure 4 Using phosphoric acid and sodium hydroxide diluted with multiple times of water, buffer solutions with pH values ​​of 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, and 8.25 were prepared. 3 mL of each of the eight PBS buffer solutions was placed in centrifuge tubes, and probe stock solution was added to each tube. Tyrosinase test solution was added to the PBS buffer solutions at different pH values, and the fluorescence intensity of the two test solutions was compared to verify the probe's resistance to pH interference.

[0044] Example 6: Molecular docking simulation test of LZD with tyrosinase

[0045] We performed molecular docking simulations. The docking results showed that there was no strong π-stacking interaction between the base pairs of TYR and the aromatic ring in LZD. Instead, the probe could be well contained within the cavity of TYR, possibly through hydrogen bonds or van der Waals interactions. The binding energy reached -6.04 kcal / mol, which also demonstrates that the interaction between LZD and mtDNA is a binding mode. Figure 5 ).

[0046] Example 7: Cytotoxicity Test

[0047] We performed dark cytotoxicity assays using the MTT (5-dimethylthiazol-2-yl-2,5-diphenyltetrazol bromide) method. Various concentrations of LZD (0 μM, 5.0 μM, 10.0 μM, 15.0 μM, 20.0 μM) were added to live HepG2 cells, and after incubation in the dark for 24 hours, the results were tested. Figure 6 As shown above, LZD has low biotoxicity and can be used in biological applications.

[0048] Example 8: Stability experiment of LZD in mitochondria

[0049] The photostability of LZD in cells is crucial because long-term monitoring is often required when studying cellular physiological processes such as apoptosis. Figure 7 In photostability experiments in HepG2 cells, the fluorescence intensity remained stable within 75 minutes, with no significant signal loss observed. This indicates that LZD exhibits good photostability and can be further tested over extended periods.

[0050] Example 9: Imaging of intracellular tyrosinases with LZD

[0051] HepG2 cells were first cultured with LZD for 0.5 hours, followed by confocal fluorescence microscopy analysis. Figure 8 As shown, fluorescence emitted by the cells can be observed in the green and red channels, respectively. Subsequently, the images obtained after adding TYR to the experimental group cells showed stronger green fluorescence and weaker red fluorescence, confirming the response of LZD to intracellular TYR.

[0052] In the endogenous TYR detection experiment, treating cells with Kojicacid for 3 hours inhibited intracellular TYR activity. Figure 9 After incubation with the probe for 0.5 h, the fluorescence intensity of the green channel in the confocal image significantly decreased, while the red channel increased. This confirms that as TYR activity is inhibited, the LZD response also weakens.

Claims

1. A dual-channel near-infrared ratiometric fluorescent probe for detecting tyrosinase, characterized in that... Its structure is as follows: 。 2. A method for preparing the dual-channel near-infrared ratiometric fluorescent probe of claim 1, characterized in that... Includes the following steps: Step 1: Mix N,N-dimethylformamide with dichloromethane, add phosphorus tribromide dropwise at 0°C, remove from ice-water bath and add cyclohexanone dropwise; after reacting for six hours, add the reaction solution dropwise to water, then neutralize with sodium carbonate, extract with water and DCM, and evaporate to dryness to obtain intermediate 1; Step 2: Add intermediate 1, 2-hydroxy-4-methoxybenzaldehyde and potassium carbonate to DMF and react for 12 h. Then extract with water and DCM, evaporate to dryness, pulp, and filter to obtain intermediate 2. Step 3: Add 3-hydroxybenzyl alcohol to DCM, add phosphorus tribromide dropwise at 0°C, react at room temperature for 2 hours, dilute DCM, extract, and rotary evaporate to obtain intermediate 3; Step 4: Add intermediate 3 and tetramethylquinoline to ethanol, reflux at 80°C for 12 h, evaporate the solvent, and separate and purify to obtain intermediate 4; Step 5: Add intermediate 2, intermediate 4 and piperidine to ethanol, reflux at 80°C for 12 h, evaporate the solvent by rotary evaporation, and slurry with ethyl acetate to obtain intermediate 5; Step 6: Add intermediate 5 and TEA to DCM, add benzoyl chloride dropwise at 0℃, react at room temperature for 8-12 h, evaporate the solvent, and separate and purify to obtain the target product LZD; The synthesis route is shown below: 。 3. The use of the dual-channel near-infrared ratiometric fluorescent probe according to claim 1, characterized in that: A tyrosinase detection reagent was prepared using the dual-channel near-infrared ratiometric fluorescent probe. The detection reagent can detect endogenous and exogenous tyrosinases in cells using dual-channel imaging.

4. The use according to claim 3, characterized in that: When tyrosinase is detected using the aforementioned detection reagent, the ratio of fluorescence intensity at 715 nm to fluorescence intensity at 510 nm in the detection system is I. 715 / I 510 There is a linear relationship between the concentration of tyrosinase and the concentration of tyrosinase.

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