Mitochondria-targeted fluorescent lightening type diarylethene organic compound and bioimaging application thereof

Abstract

The invention discloses a mitochondrial targeting fluorescence lightening type diarylethene organic compound and application thereof in biological imaging, the mitochondrial targeting fluorescence lightening type diarylethene organic compound has a structure as shown in a general formula (1), and in the general formula (1), R1 represents an alkyl chain, and R2 represents a mitochondrial targeting group. All the fluorescent lighting type diarylethene photochromic materials disclosed by the invention have good biocompatibility, and accurate mitochondrial targeting can be realized by all the fluorescent lighting type diarylethene photochromic materials.

Description

Technical Field

[0001] This invention relates to photochromic materials, and particularly to mitochondrial-targeted fluorescently illuminated diarylethylene photochromic materials and their bioimaging applications. Background Technology

[0002] Bioimaging technology, as a core tool for life science research, disease diagnosis, and drug development, can intuitively present dynamic processes at the molecular level within organisms, providing crucial support for understanding the mechanisms of life activities and early disease screening. Among these, fluorescence imaging technology, with its advantages of high sensitivity, high spatiotemporal resolution, ease of operation, and minimal damage to biological samples, has become one of the most widely used imaging methods in the biomedical field. Fluorescent probes, as a core component of fluorescence imaging technology, directly determine image quality and application effectiveness. Therefore, developing fluorescent probes with high selectivity, high stability, low biotoxicity, and targeting specificity is currently a research hotspot and core requirement in the field of bioimaging.

[0003] Among numerous biological targets, mitochondria, as the cell's "energy factory," participate in key life processes such as cellular respiration, energy metabolism, signal transduction, and apoptosis. Abnormalities in their structure and function are closely related to the occurrence and development of various diseases, such as cancer, neurodegenerative diseases, and cardiovascular diseases. Therefore, achieving precise targeted imaging of mitochondria and real-time monitoring of their dynamic changes and microenvironmental parameters (such as membrane potential and reactive oxygen species levels) is of great significance for in-depth research into mitochondrial-related physiological and pathological mechanisms and the development of novel disease diagnostic methods.

[0004] Diarylethylene compounds are a class of photochromic organic functional materials whose molecular structures undergo reversible cyclization / ring-opening isomerization reactions under ultraviolet and visible light irradiation, accompanied by significant changes in optical properties such as absorption and fluorescence spectra. These compounds possess advantages such as high photostability, strong resistance to photobleaching, fast response speed, and long cycle life, and have been widely used in fields such as optical switches, fluorescence sensors, and super-resolution imaging.

[0005] Introducing the diarylethene structure into fluorescent probe design allows for the construction of fluorescently lit photoresponsive probes: In the absence of UV irradiation, the probe is in an open-ring state, exhibiting a weak fluorescence signal; upon UV excitation, the probe undergoes cyclization and isomerization, resulting in a significant enhancement of the fluorescence signal (i.e., the "lighting-up" effect). This characteristic effectively reduces imaging background noise and improves detection sensitivity. Furthermore, the molecular structure of diarylethene compounds can be flexibly controlled through chemical modification, providing possibilities for achieving mitochondrial targeting and multi-response functional integration. Summary of the Invention

[0006] Although diarylethylene compounds have shown great potential in the field of photoresponsive fluorescent materials, research on their application in mitochondrial-targeted bioimaging is still in its early stages, with significant gaps in existing technologies:

[0007] (1) There is a lack of design strategies for diarylethylene compounds that combine mitochondrial targeting and photochromic fluorescence illumination. Most existing diarylethylene derivatives do not have clear biological targeting functions, making it difficult to achieve precise localization imaging of mitochondria.

[0008] (2) Existing mitochondrial targeting probes have many compatibility issues when combined with diarylethene structures. For example, the introduction of the targeting group may destroy the photochromic properties of diarylethene, or the imbalance between the lipid solubility and water solubility of the probe molecule may lead to a decrease in biocompatibility.

[0009] (3) No diarylethene-based bioimaging probes have yet been developed that can simultaneously meet the requirements of high targeting, high fluorescence illumination efficiency, excellent biocompatibility and responsiveness to the mitochondrial microenvironment.

[0010] Therefore, in order to address the shortcomings of existing bioimaging probes and the gap in the application of diarylethylene compounds in the biomedical field, it is of great scientific significance and practical application value to develop a mitochondrial-targeting fluorescently lit diarylethylene organic compound to achieve high-sensitivity, low-background, long-term dynamic imaging of mitochondria and apply it to the study of mitochondrial-related physiological and pathological conditions and disease diagnosis.

[0011] The technical solution of the present invention is as follows:

[0012] A mitochondrial-targeted fluorescently lit diarylethylene organic compound, characterized by having a structure as shown in general formula (1):

[0013]

[0014] In general formula (1), R1 represents a C1 to C6 alkyl group;

[0015] In general formula (1), R2 represents the mitochondrial targeting group.

[0016] This invention utilizes a strategy of attaching a targeting group to a bridging thiophene on an existing fluorescently lit diarylethene molecular backbone. This strategy can both improve the absorption intensity in the closed-ring molecular structure and achieve precise mitochondrial targeting, thereby obtaining a diarylethene photochromic material that can achieve visible high fluorescence quantum yield and fluorescently lit mitochondrial targeting.

[0017] Preferably, in the mitochondrial-targeted fluorescently lit diarylethene organic compound, R1 in general formula (1) represents one of the following structures:

[0018] -CH3, -C2H5, -C3H7, -C4H9, -C5H 11 .

[0019] Preferably, in the mitochondrial-targeted fluorescently lit diarylethene organic compound, R2 in general formula (1) represents one of the following structures:

[0020] Furthermore, this compound contains negatively charged ions that balance the charge; specifically, the negative ion is Br. - .

[0021] Preferably, the mitochondrial-targeted fluorescently illuminated diarylethylene organic compound is one of the following specific compounds:

[0022]

[0023]

[0024]

[0025] Preferably, the diarylethylene organic compound 11 according to claim 4 has the following structural formula:

[0026]

[0027] This invention provides an application of the aforementioned mitochondrial-targeted fluorescent brightening diarylethylene in the field of bioimaging.

[0028] Preferably, the mitochondrial-targeted fluorescently lit diarylethylene is used to prepare a fluorescent probe.

[0029] As a further preferred embodiment, the fluorescent probe is used for selective imaging of mitochondria within cells.

[0030] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0031] This invention addresses the poor compatibility between the target group and the diarylethylene structure in existing technologies by precisely chemically modifying the parent structure of diarylethylene and introducing an optimized mitochondrial targeting group. The target group and the diarylethylene parent structure are covalently bound via a flexible linker, preserving the target group's specific recognition ability of mitochondrial membrane potential without compromising the photochromic core properties of diarylethylene. Experimental verification shows that the compound of this invention achieves a mitochondrial enrichment efficiency of over 90% in cells, and a colocalization coefficient with the commercial mitochondrial probe Mito Tracker Green is higher than 0.95, significantly outperforming existing non-targeted diarylethylene derivatives and some targeted fluorescent probes. This effectively reduces imaging background noise caused by non-specific binding, achieving precise mitochondrial localization imaging.

[0032] This invention optimizes the molecular structure to balance the lipid and water solubility of the compound, avoiding the intracellular aggregation and precipitation problems caused by excessive lipid solubility in existing probes. Cytotoxicity assays (MTT assay) show that at effective imaging concentrations (0.1-5 μM), the compound exhibits survival rates exceeding 90% against human hepatocellular carcinoma cells (HepG2) and human neuroblastoma cells (SH-SY5Y), significantly lower than the cytotoxicity of some existing mitochondrial probes. Furthermore, the compound demonstrates good stability within the physiological pH range (pH 6.5-7.8) without significant fluorescence quenching, enabling safe and stable imaging applications in multi-scale biological samples, including cells, tissue sections, and live small animals, thus expanding the applicable scenarios for mitochondrial-targeted fluorescent probes. Attached Figure Description

[0033] Figure 1 This is a fluorescence confocal imaging image of compound 11. Detailed Implementation

[0034] The present invention will now be described in detail with reference to the embodiments.

[0035] Example: Synthesis of Compound 11:

[0036] Synthesis route:

[0037]

[0038] In a 50 mL two-necked flask, 0.30 mmol of starter 1, 0.40 mmol of 4-tert-butoxycarbonylphenylboronic acid, and 10 mL of tetrahydrofuran solution were added sequentially and stirred until dissolved. Then, 5 mL of saturated K₂CO₃ solution and 0.08 mmol of Pd(PPh₃)₄ were added. The solution was degassed with nitrogen for approximately 15 minutes and then refluxed for 8 hours. After cooling the reaction mixture to room temperature, it was extracted with ethyl acetate. The combined organic phases were dried over anhydrous Na₂SO₄ and then evaporated to dryness. Purification by silica gel column chromatography (DCM / hexane = 1:3) yielded product 1 (0.285 mmol, 0.176 g, 95% yield). 1 H NMR (400MHz, CDCl3): δ[ppm]1.42(s,9H),1.99-1.55(t,6H),6.63(s,1H),7.30-7.63(m,4H),7.71-7.79(m,4H),7.90-7.92(m,2H),7.96(m,2H); 13 C NMR (100MHz, CDCl3): δ[ppm]164.6,143.4,138.9,138.0,135.3,133.6,132.9,13 0.6,130.1,128.9,128.1,127.4,127.1,122.9,81.7,28.7,8.61,8.33.HRMS[M+H] + calcd.for C 33 H 28 O6S3:616.1048, found 616.1053.

[0039] In a 50 mL double-necked flask, 0.20 mmol of product 1 was added, followed by 10 mL of trifluoroacetic acid and dichloromethane. The reaction was stopped after 3 h, and the solution was evaporated to dryness to obtain crude product 2 for further reaction.

[0040] 0.10 mmol of product 2, 0.20 mmol of (2-aminoethyl)triphenylphosphine bromide (109 mg), 0.20 mmol of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and 0.20 mmol of N,N-diisopropylethylamine were dissolved in N,N-dimethylformamide (DMF, 3 mL), and the mixture was stirred at room temperature for 5 hours. After the reaction was completed, the solvent was removed, the crude product was extracted with dichloromethane, washed with water, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel rapid column chromatography (DCM / hexane = 1:1) to give compound 11 (0.07 mmol, 38 mg, yield 70%).

[0041] 1H NMR (400MHz, CDCl3): δ[ppm]1.99-1.25(t,6H),2.22-2.27(m,2H),3.21-3.28(m,2H),6.65(s, 1H),7.30-7.39(m,15H),7.51-7.59(m,2H),7.69-7.78(m,6H),7.95-8.02(m,4H),8.48(s,1H); 13 C NMR (100MHz, CDCl3): δ[ppm]167.5,143.4,138.9,137.8,137.1,135.3,133.6,13 2.9,130.6,128.9,127.6,127.4,132.2,117.9,29.1,27.5,8.62,8.34.HRMS[M+H] + calcd.for C 49 H 39 NO5PS3:848.1722, found 848.1743.

[0042] The vertical absorption energy, oscillator strength, HOMO / LUMO energy level, and band gap between HOMO and LUMO were also quantitatively calculated for the open-ring and closed-ring forms of compound 11 of the present invention. The maximum emission, fluorescence quantum yield, fluorescence lifetime, and S1 energy level were tested, and the results are shown in Table 1.

[0043] Table 1: Theoretical calculations and experimental data for compound 11

[0044]

[0045]

[0046] The above experimental results show that the closed-ring form of compound 11 of this application has extremely high fluorescence quantum efficiency and can successfully achieve visible light-driven cyclization and high fluorescence quantum yield, which has good prospects for bioimaging applications.

[0047] Application Example 1

[0048] Inspired by its excellent optical properties, Example 1 systematically evaluated the performance of compound 11 in detecting live cells. Cytotoxicity is a key consideration for its use in live systems, and we evaluated it in HeLa cells using the MTT assay. The results showed that low micromolar concentrations of compound 11 exhibited virtually no cytotoxicity within 24 hours, making it suitable for biological applications. Subsequently, we conducted live-cell imaging experiments: HeLa cells stained with compound 11 showed clear fluorescence signals, indicating that the probe has excellent cell membrane permeability. Co-staining with mitochondrial tracker green further validated its mitochondrial targeting characteristics—the superposition of red and green fluorescence resulted in a yellow color, and quantitative analysis showed a Pearson correlation coefficient as high as 0.96 (see results in [link to study]). Figure 1 ).

[0049] While the invention has been disclosed through embodiments and preferred embodiments, it should be understood that the invention is not limited to the disclosed embodiments. Rather, those skilled in the art will appreciate that it is intended to cover various variations and similar arrangements. Therefore, the scope of the appended claims should be consistent with the broadest interpretation to cover all such variations and similar arrangements.

Claims

1. A mitochondrial-targeted fluorescently lit diarylethylene organic compound, characterized in that, The structure is shown in general formula (1): In general formula (1), R1 represents a C1 to C6 alkyl group; In general formula (1), R2 represents the mitochondrial targeting group.

2. The mitochondrial-targeted fluorescently lit diarylethylene organic compound according to claim 1, characterized in that, In general formula (1), R1 represents one of the following structures: -CH3, -C2H5, -C3H7, -C4H9, -C5H 11 。 3. The mitochondrial-targeted fluorescently lit diarylethylene organic compound according to claim 1, characterized in that, In general formula (1), R2 represents one of the following structures:

4. The mitochondrial-targeted fluorescently lit diarylethylene organic compound according to claim 1, characterized in that, It is one of the following specific compounds:

5. The mitochondrial-targeted fluorescently illuminated diarylethylene according to claim 4, characterized in that, The diarylethene organic compound 11 has the following structural formula:

6. The application of a mitochondrial-targeted fluorescent brightening diarylethylene as described in any one of claims 1 to 5 in the field of bioimaging.

7. The application according to claim 6, characterized in that, The mitochondrial-targeted fluorescent brightening diarylethylene is used to prepare fluorescent probes.

8. The application according to claim 7, characterized in that, The fluorescent probe is used for selective imaging of mitochondria within cells.

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