A lysosome-targeted super-sensitive polarity-responsive fluorescent probe and a preparation method thereof

Abstract

The application discloses a lysosome-targeting super-sensitivity polarity-responsive fluorescent probe and a preparation method thereof. The application takes phenothiazine as an electron donor, furan-olefin as a pi conjugated bridge, and ethyl cyanoacetate as an electron-withdrawing acceptor to prepare a phenothiazine derivative fluorescent probe with an A-pi-D-pi-A structure. The probe has a simple synthesis method, near-infrared emission, a large stokes shift and high quantum yield, and shows super-sensitivity response to solvent polarity. The probe realizes excellent lysosome targeting ability through ethyl ester modification, and can accurately monitor lysosome polarity changes caused by factors such as lipid overload in living cells. The fluorescent probe has wide application prospects in cell biology, pathological mechanism research and drug screening, especially in researches on diseases related to lysosome dysfunction.

Description

Technical Field

[0001] This invention relates to an ultrasensitive polar-responsive fluorescent probe targeting lysosomes and its preparation method. Specifically, it belongs to the field of fluorescence sensing and bioimaging technology. Background Technology

[0002] Polarity, as an important physicochemical parameter characterizing the microenvironment of matter, plays a crucial role in cellular physiological and pathological processes. Abnormal changes in cellular microenvironment polarity are closely related to the occurrence and development of various diseases, such as inflammation, cancer, and lipid metabolism disorders. Therefore, developing fluorescent probes capable of real-time, sensitive, and visual monitoring of intracellular polarity is of great significance for elucidating disease mechanisms and early diagnosis.

[0003] Lysosomes are important metabolic and degradation centers within cells, and changes in their microenvironment (especially polarity) directly affect their function. For example, under pathological conditions such as lipid overload (e.g., non-alcoholic fatty liver disease), lysosomal polarity fluctuates significantly. Currently, although polarity-responsive fluorescent probes have been reported, such as probes based on intramolecular charge transfer (ICT) mechanisms, some shortcomings remain: some probes have low sensitivity and are difficult to capture minute polarity changes; most probes emit visible light, have weak tissue penetration, and are easily interfered with by biological background fluorescence; polar probes that combine near-infrared emission, high sensitivity, and the ability to target specific organelles (such as lysosomes) are still relatively scarce.

[0004] Therefore, developing a fluorescent probe with near-infrared emission, ultrasensitive polar response, and the ability to specifically target lysosomes is of great value for a deeper understanding of lysosomal function and the pathological mechanisms of related diseases. Summary of the Invention

[0005] To address the aforementioned problems and shortcomings of existing technologies, the present invention aims to provide an ultrasensitive polar-responsive fluorescent probe with lysosomal targeting capability, its preparation method, and its applications. This probe uses phenothiazine as an electron donor, constructs an A-π-D-π-A type structure to achieve near-infrared emission and ultrasensitive polar response, and endows it with lysosomal targeting properties by introducing an ethyl ester group.

[0006] The structural formula of the ultrasensitive polar-responsive fluorescent probe targeting lysosomes of the present invention is shown in formula a: ; The preparation method of a highly sensitive polar-responsive fluorescent probe targeting lysosomes is as follows: Step 1: Under argon protection, 3,7-dibromo-10-octylphenthiazide and 5-formylfuran-2-boronic acid were subjected to a Suzuki coupling reaction in tetrahydrofuran in the presence of tetratriphenylphosphine palladium to obtain the intermediate 3,7-bis(5-formylfuran-2-yl)-10-octylphenthiazide. Step 2: The intermediate obtained in Step 1 was reacted with ethyl cyanoacetate in ethanol under pyridine catalysis to undergo a Knoevenagel condensation reaction. After the reaction was completed, the sample was separated and purified to obtain an ultrasensitive polar-responsive fluorescent probe.

[0007] The reaction equation for this invention is as follows: ; Beneficial effects of this invention: 1. This invention employs a novel A-π-D-π-A molecular design strategy, using phenothiazine as the donor, furan ring and olefin double bond structure as bridging units, and ethyl cyanoacetate as the acceptor. This enhances the intramolecular charge transfer (ICT) effect, achieving red / near-infrared emission above 628 nm and a large Stokes shift greater than 150 nm. This effectively avoids interference from biological background fluorescence and improves the signal-to-noise ratio of biological imaging.

[0008] 2. This invention exhibits an ultrasensitive linear response to solvent polarity. The quantum yield reaches as high as 72% in low-polarity environments, while fluorescence quenches rapidly in high-polarity environments. It is extremely sensitive to trace amounts of highly polar solvents (such as DMSO and H2O), and the emission wavelength shows a good linear relationship with the solvent polarity parameter.

[0009] 3. This invention employs a novel lysosome-targeting strategy. Unlike conventional morpholine ring targeting groups, it achieves specific targeting of lysosomes through the introduction of ethyl ester groups, with a co-localization coefficient reaching 0.92. It exhibits extremely low cytotoxicity, maintaining cell viability above 95% at commonly used concentrations of 0-20 μM. It has been successfully used to monitor the decrease in lysosomal polarity in an oleic acid-induced lipid overload model, with significantly enhanced fluorescence intensity and luminescent area, providing a new visualization tool for studying the pathological mechanisms of lipid metabolism disorders (such as non-alcoholic fatty liver disease). Attached Figure Description

[0010] Figure 1 Example 1 of the present invention describes the preparation of compound b. 1 HNMR; Figure 2 Example 2 of the present invention: Preparation of compound a 1 HNMR; Figure 3 Fluorescence emission spectra of compound a in different solvents in Example 3 of this invention; Figure 4The fluorescence emission spectrum of compound a in the THF / Diox mixed system in Example 3 of this invention; Figure 5 The fluorescence emission spectrum of compound a in the DMSO / Diox mixed system in Example 3 of this invention; Figure 6 The cytotoxicity of compound a in Example 5 of this invention; Figure 7 : Cell confocal analysis results of compound a in Example 6 of this invention; Figure 8 The structural formula of the ultrasensitive polar-responsive fluorescent probe targeting lysosomes of this invention. Detailed Implementation

[0011] Example 1 ; Accurately weigh 3,7-dibromo-10-octylphenthiazide (2.00 g, 4.28 mmol), 5-formyl-2-furanboronic acid (1.50 g, 10.7 mmol), triphenylphosphine palladium (0.63 g, 0.85 mmol), and potassium carbonate (2.37 g, 17.12 mmol), and dissolve them in a flask with a side-mounted bowl and Diox / H₂O (3 / 1, V / V). React under argon protection for 24 h, extract with ethyl acetate, and purify by silica gel column chromatography. Eluent with PE / EA = 5 / 1 yielded 1.38 g of an orange-yellow viscous solid with a yield of 65%. This yielded intermediate b, with the structure 3,7-bis(5-formylfuran-2-yl)-10-octylphenthiazide.

[0012] Example 2 ; Intermediate b (500 mg, 0.4 mmol), ethyl cyanoacetate (255 mg, 1.2 mmol), pyridine (0.1 ml), and dichloromethane (20 ml) were added to a single-necked flask. The reaction was carried out at room temperature for 12 h. After extraction with deionized water (20 ml × 3) and drying of the crude product, the product was recrystallized from dichloromethane:n-hexane (5 ml:25 ml) to give 220 mg of black solid, with a yield of 78%. This yielded the target product a.

[0013] Example 3 Weigh an appropriate amount of solid a and dissolve it in toluene, 1,4-dioxane, tetrahydrofuran, dichloromethane, and dimethyl sulfoxide, respectively, to prepare 10 μM test solutions. Measure its UV absorption and fluorescence emission spectra using a UV spectrophotometer and a fluorescence spectrophotometer, and simultaneously photograph its fluorescence under UV light. a exhibits strong red emission in low-polarity toluene and 1,4-dioxane, while the emission peak shows a significant red shift and the fluorescence emission intensity decreases considerably in other medium- to high-polarity solvents.

[0014] Example 4 Dissolving 'a' in mixed solvents of varying proportions, the fluorescence emission spectra were measured and images were captured. In the THF / Diox mixed solvent system, fluorescence emission gradually quenched with increasing solvent polarity, and the emission wavelength of M3 showed a good linear relationship with the solvent polarity parameter (Δf). In the DMSO / Diox system, adding only 1% volume fraction of DMSO was sufficient to quench approximately 50% of the fluorescence intensity.

[0015] Example 5 The cytotoxicity of a to HepG2 cells was assessed using the CCK-8 assay. After co-incubating cells with probe solutions of different concentrations (0.05, 0.1, 0.5, 1, 2, 5, 10, 20, 40 μM) for 24 hours, cell viability remained above 90% in all concentration groups, indicating that a has good biocompatibility and low cytotoxicity.

[0016] Example 6 HepG2 cells were divided into two groups. The experimental group was pretreated with 200 μM oleic acid for 24 hours to induce lipid overload, while the control group received no treatment. Both groups of cells were then co-incubated with a probe (5 μM) and LysoTracker Green. Imaging results showed that the probe fluorescence was relatively weak in the untreated group, with a co-localization coefficient of 0.73 with LysoTracker Green. In the oleic acid-treated group, the red fluorescence of the probe was significantly enhanced, and the co-localization coefficient with LysoTracker Green increased to 0.92, while there was no co-localization with the lipid droplet dye BODIPY 495 / 503 (Pearson coefficient 0.12). Quantitative analysis using ImageJ software showed that after oleic acid treatment, the intracellular fluorescence luminescence area of ​​the probe was approximately 1.5 times that of the control group, and the average fluorescence intensity was approximately 1.8 times that of the control group. These results confirm that the probe of this invention can accurately report the process of decreased lysosomal polarity by enhancing the fluorescence signal and expanding the luminescence area.

Claims

1. A highly sensitive polar-responsive fluorescent probe targeting lysosomes, characterized in that: The structure of the fluorescent probe is shown in formula a: 。 2. A method for preparing a highly sensitive polar-responsive fluorescent probe targeting lysosomes, characterized in that: The preparation method steps are as follows: Step 1: Under argon protection, 3,7-dibromo-10-octylphenthiazide and 5-formylfuran-2-boronic acid were subjected to a Suzuki coupling reaction in tetrahydrofuran in the presence of tetratriphenylphosphine palladium to obtain the intermediate 3,7-bis(5-formylfuran-2-yl)-10-octylphenthiazide. Step 2: The intermediate obtained in Step 1 was reacted with ethyl cyanoacetate in ethanol under pyridine catalysis to undergo a Knoevenagel condensation reaction. After the reaction was completed, the sample was separated and purified to obtain an ultrasensitive polar-responsive fluorescent probe.

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