Preparation method and application of viscosity-responsive lysosome fluorescent probe
A TICT-based near-infrared fluorescent probe addresses the challenge of monitoring lysosomal viscosity changes with high sensitivity and stability, enabling real-time disease diagnosis and treatment.
Patent Information
- Application Number
- CN202411313906.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-20
- Publication Date
- 2024-12-27
AI Technical Summary
Existing fluorescent probes lack sensitivity and stability for real-time monitoring of lysosomal viscosity changes, which are crucial for understanding and diagnosing diseases like cancer and neurodegenerative diseases.
A novel near-infrared fluorescent probe based on the twisted intramolecular charge transfer (TICT) effect is developed, providing high sensitivity and stability in lysosomal environments, with a specific preparation method involving dihydroisophorone and 5-morpholinothiophene-2-carboxaldehyde, refluxing in 1,4-dioxane with glacial acetic acid and piperidine, followed by purification.
The probe effectively monitors dynamic lysosomal viscosity changes with significant fluorescence signal variations, offering a new tool for early disease diagnosis and treatment, particularly in cancer and neurodegenerative diseases.
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Figure CN119192130A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of chemical medicine, and in particular to a preparation method and application of a viscosity-responsive lysosomal fluorescent probe. Background Art
[0002] Lysosomes are important organelles in cells, responsible for degrading and recycling intracellular waste, damaged organelles, and foreign substances. Abnormal lysosomal function is closely related to a variety of diseases, such as cancer, neurodegenerative diseases, cardiovascular diseases, and metabolic diseases. Therefore, real-time monitoring and evaluation of changes in the lysosomal environment are crucial for studying the mechanisms of these diseases and developing new diagnostic and therapeutic methods. Fluorescent probe technology has become one of the main means of studying changes in the intracellular microenvironment due to its advantages such as high sensitivity, real-time, and non-invasiveness. Viscosity-responsive fluorescent probes are a type of probe that can reflect changes in environmental viscosity through changes in fluorescence signals. This type of probe is usually based on the twisted intramolecular charge transfer (TICT) quenching mechanism. The molecular conformation or motion state of the probe molecule changes in different viscosity environments, resulting in changes in the fluorescence signal. The development of new viscosity-responsive lysosomal fluorescent probes will not only help to deeply understand the role of lysosomes in various physiological and pathological processes, but also provide new technical means for the early diagnosis and treatment of lysosome-related diseases. Especially in the study of major diseases such as cancer and neurodegenerative diseases, viscosity-responsive lysosomal fluorescent probes have broad application prospects. Therefore, a preparation method of a viscosity-responsive lysosomal fluorescent probe and its use are disclosed for monitoring the dynamic changes of lysosomal viscosity. This project is supported by the Qinghai Province Applied Basic Research Project (2024-ZJ-701). Summary of the invention
[0003] Based on the current status of the above-mentioned prior art research, the present invention provides a method for preparing a viscosity-responsive lysosomal fluorescent probe and its use. The fluorescent probe of the present invention has a sensitive fluorescent response to changes in lysosomal viscosity, can maintain high stability and sensitivity in the lysosomal environment, and reflects the dynamic changes in lysosomal viscosity through changes in fluorescent signals.
[0004] Specifically, the present invention provides a fluorescent probe, the structure of which is shown in the following formula (I):
[0005]
[0006] The present invention is a novel near-infrared fluorescent probe based on the twisted intramolecular charge transfer effect (TICT effect). The probe has good lysosomal targeting and shows significant fluorescence signal changes under different viscosity environments. It provides a new tool for real-time monitoring of viscosity changes in lysosomes. The probe has broad application prospects in the research of cancer, neurodegenerative diseases and metabolic diseases, and is expected to provide new ideas and methods for the early diagnosis and treatment of related diseases.
[0007] The specific preparation method of the fluorescent probe of the present invention is as follows: dihydroisophorone (0.558mmol, 103mg) and 5-morpholinothiophene-2-carboxaldehyde (0.507mmol, 100mg) are added to a 100mL round-bottom flask, and then 1,4-dioxane (5mL), glacial acetic acid (HAC, 0.4mL) and piperidine (0.4mL) are added in sequence. The mixture is refluxed at 130°C for 2h. The mixture is cooled to room temperature, extracted with dichloromethane, and washed with brine. The combined organic phase is dried over anhydrous sodium sulfate. The solvent is evaporated under reduced pressure. The crude product is purified by silica gel column chromatography (PE / EA=4 / 1). BRIEF DESCRIPTION OF THE DRAWINGS
[0008] Figure 1 It is a schematic diagram of the fluorescence spectrum of Example 1 in a water-glycerol mixture with a glycerol content of 0% to 90%.
[0009] Figure 2 It is a schematic diagram of the cytotoxicity results of Example 1 on HeLa cells (a) and HepG2 cells (b).
[0010] Figure 3 This is a schematic diagram of the laser fluorescence confocal image of HepG2 cells in Example 1.
[0011] Example 1
[0012] (E)-2-(5,5-dimethyl-3-(2-(5-morpholinothiophen-2-yl)vinyl)cyclohex-2-en-1-ylidene)malononitril e: 1 H NMR (600MHz, CDCl3) δ7.09 (d, J = 15.4Hz, 1H), 6.95 (d, J = 4.1Hz, 1H), 6.67 (s, 1H), 6.40 (d, J = 15.4Hz, 1H), 6.03 (d ,J=4.1Hz,1H),3.85(dd,J=5.8,4.0Hz,4H),3.25(dd,J=6.0,3.8Hz,4H),2.54(s,2H),2.38(s,2H),1.04(s,6H). 13CNMR(151MHz,CDCl3)δ168.70,162.05,154.66,132.65,131.35,127.66,123.52,1 20.95,114.40,113.61,105.06,66.01,50.10,42.92,39.20,31.96,29.71,28.05.
[0013] Experimental Example 1: Fluorescence response experiment of Example 1 in glycerol-PBS mixtures with different ratios.
[0014] Experimental method: The compound in Example 1 was added to 2 mL of solutions of different viscosities consisting of PBS (pH=7.4) and glycerol to a final concentration of 10 μM. Fluorescence spectra were measured in these solutions.
[0015] Experimental results: The results are as follows Figure 1 As shown, the results show that the fluorescence intensity of Example 1 increases with the increase of viscosity. Experimental Example 2: Cytotoxicity test of Example 1 on HeLa cells and HepG2 cells Experimental method: 1×10 4 HeLa cells and HepG2 cells were treated with the compounds in Example 1 for 24 hours, and the cytotoxicity was determined by MTT method.
[0016] Experimental results: The results are as follows Figure 2 As shown, the results show that Example 1 has less toxicity to HeLa cells and HepG2 cells, and the survival rate remains above 87% after incubation with the cells.
[0017] Experimental Example 3: Fluorescence intensity changes of Example 1 under different cell viscosities Experimental method: Dexamethasone (DXM, 20 μM) and lipopolysaccharide (LPS, 20 μM) were added to the HepG2 cell culture medium. After incubation for 30 minutes and washing 3 times with PBS, Example 1 was added and incubated for another 30 minutes. Confocal fluorescence imaging of HepG2 cells was performed using bright field and red fluorescence channels.
[0018] Experimental results: The results are as follows Figure 3 As shown, the results show that the fluorescence intensity of Example 1 is different in different cell viscosity microenvironments, and the higher the viscosity, the stronger the fluorescence intensity.
Claims
1. A dihydroisophorone derivative, characterized in that: Its structure is shown in the following formula (I):
2. The fluorescent probe according to claim 1, characterized in that The compound is shown below: #imgpt2# 3. A new use of the fluorescent probe as claimed in claim 1, characterized in that: It can maintain high stability and sensitivity in the lysosomal environment and reflect the dynamic changes of lysosomal viscosity through changes in fluorescence signals.