Mitochondrial targeting fluorescent dye as well as synthesis and application thereof

Abstract

The invention belongs to the field of fluorescent dyes and biological imaging, and particularly provides a mitochondrial targeting fluorescent dye as well as synthesis and application thereof. The structural formula of the mitochondrial targeting fluorescent dye is shown as a formula (I). The compound is synthesized by one-step heating reaction of benzothiazole-2-acetonitrile and Fisher's aldehyde under the action of alkali. The mitochondrial targeting fluorescent dye has good cell membrane penetrability, can be rapidly taken by cells, and is good in mitochondrial targeting, high in light stability and low in toxicity to the cells. Meanwhile, the preparation process is simple, the cost is low, clear and long-time fluorescence imaging analysis and dynamic fluorescence monitoring on mitochondria in cells can be realized, and the fluorescent probe has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent dyes and bioimaging, specifically relating to a mitochondrial-targeting fluorescent dye and its synthesis and application. Background Technology

[0002] Mitochondria, as the "energy factories" of eukaryotic cells, are not only the main site of adenosine triphosphate (ATP) synthesis, but also deeply involved in a series of vital life activities such as cellular metabolism, calcium ion homeostasis maintenance, reactive oxygen species (ROS) generation, and ferroptosis. Mitochondrial dysfunction is closely related to the development and progression of neurodegenerative diseases (such as Alzheimer's and Parkinson's), cardiovascular diseases, metabolic diseases (such as diabetes), and various cancers. Developing efficient mitochondrial imaging tools is of great significance for assessing cellular energy metabolism levels and health status, revealing physiological processes such as cell differentiation, proliferation, autophagy, and programmed cell death, as well as for early diagnosis, drug screening, and efficacy evaluation of diseases.

[0003] Fluorescence imaging technology, with its high sensitivity, high spatiotemporal resolution, ease of operation, and non-destructive real-time monitoring, has become an indispensable tool in biological and medical research. Using mitochondrial-targeting fluorescent dyes, researchers can observe the dynamic network structure, morphological changes, membrane potential fluctuations, and intracellular distribution and migration of mitochondria in real time and visually in living cells. This non-invasive imaging method can not only clearly reveal the behavior of mitochondria in key life processes such as cell metabolism, division and fusion, and apoptosis, but also be used to assess cell viability and pathological states.

[0004] Currently, numerous fluorescent dyes for labeling mitochondria have been reported and used. However, existing commercially available mitochondrial dyes (such as Rhodamine 123 and the MitoTracker series) have significant drawbacks: First, their synthesis and purification processes are complex and costly; second, they have poor photostability and are prone to photobleaching during long-term imaging, making it difficult to achieve dynamic and long-term tracking of mitochondria. Furthermore, some mitochondrial dyes are slowly taken up by cells, requiring prolonged incubation, which not only affects diagnostic efficiency but may also interfere with normal cellular function. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a mitochondrial-targeting fluorescent dye, its synthesis and application, for fluorescence imaging analysis and dynamic fluorescence monitoring of mitochondria in cells.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A mitochondrial-targeting fluorescent dye, the structural formula of which is shown in formula (I): Formula (I).

[0007] The above-mentioned method for synthesizing mitochondrial-targeting fluorescent dyes includes the following reaction steps: Benzothiazole-2-acetonitrile and Fisher's aldehyde are added to an organic solvent and heated under the action of an alkali to obtain the fluorescent dye shown in formula (I).

[0008] Furthermore, the molar ratio of benzothiazole-2-acetonitrile to Fisher's aldehyde is 1:(1~4).

[0009] Furthermore, the organic solvent is at least one selected from methanol, ethanol, acetonitrile, acetone, 1,4-dioxane, tetrahydrofuran, or N,N-dimethylformamide.

[0010] Furthermore, the base is at least one selected from cesium carbonate, potassium carbonate, sodium carbonate, sodium bicarbonate, pyridine, piperazine, triethylamine, or N,N-dimethylaminopyridine.

[0011] Furthermore, the molar ratio of the benzothiazole-2-acetonitrile to the base is 1:(1~4).

[0012] Furthermore, the heating temperature is 20℃-60℃; the reaction time is 4-12 hours.

[0013] The present invention also provides the application of the above-mentioned mitochondrial-targeting fluorescent dye as a mitochondrial fluorescent probe.

[0014] The present invention also provides the application of the above-mentioned mitochondrial-targeting fluorescent dyes in intracellular mitochondrial fluorescence imaging and morphological monitoring for non-disease diagnosis and treatment purposes.

[0015] This invention provides a mitochondrial-targeting fluorescent dye that enables clear, long-term fluorescence imaging analysis and dynamic fluorescence monitoring of mitochondria in cells. Specific advantages are as follows: (1) It has good cell membrane permeability and can be rapidly taken up by cells. After adding the fluorescent dye of the present invention to the cell culture medium, a fluorescent signal appears in the cell after 10 seconds and reaches a stable level after 50 seconds. (2) The mitochondria have good targeting and strong photostability. After scanning 30 times under a confocal microscope, the fluorescence signal of the fluorescent dye of the present invention in the cell did not decrease significantly. (3) It has good safety. The fluorescent dye of the present invention has low toxicity to cells and will not affect cell activity during fluorescence imaging or dynamic monitoring. (4) The preparation process is simple, requiring only one-step synthesis reaction, easy to separate and purify, and the raw materials are cheap and readily available, resulting in low cost. Attached Figure Description

[0016] Figure 1 The mitochondrial-targeting fluorescent dye of the present invention1 H NMR spectrum.

[0017] Figure 2 The mitochondrial-targeting fluorescent dye of the present invention 13 C10 NMR spectrum.

[0018] Figure 3 This image shows the co-localization imaging of the mitochondrial-targeting fluorescent dye of the present invention and the commercially available mitochondrial fluorescent dye Mito-red. In Figure A, the mitochondrial-targeting fluorescent dye of the present invention is shown; in Figure B, the commercially available mitochondrial fluorescent dye Mito-red is shown; in Figure C, the overlap is shown; and in Figure D, the co-localization coefficient is shown.

[0019] Figure 4 Fluorescence imaging of HeLa cells for the uptake of the mitochondrial-targeted fluorescent dye of this invention.

[0020] Figure 5 This is a comparison of the photostability of the mitochondrial-targeting fluorescent dye of the present invention with that of the commercially available mitochondrial fluorescent dye Mito-red in cells.

[0021] Figure 6 This invention provides a cytotoxicity evaluation of the mitochondrial-targeting fluorescent dye.

[0022] Figure 7 This invention relates to the application of mitochondrial-targeting fluorescent dyes in the dynamic monitoring of mitochondrial morphological changes during epirastine-induced ferroptosis. Detailed Implementation

[0023] The present invention will be further described below with reference to the embodiments and accompanying drawings. The embodiments described below are for illustrative purposes only and do not limit the scope of protection of the present invention in any way.

[0024] Example 1 Synthesis of mitochondrial-targeting fluorescent dyes Benzothiazole-2-acetonitrile (0.35 g, 2.0 mmol), Fisher's aldehyde (0.40 g, 2.0 mmol), and anhydrous piperazine (0.16 g, 2.0 mmol) were added to methanol (40 mL), heated to 40 °C, and reacted for 8 hours, then allowed to cool naturally to room temperature. The reaction solution was filtered, and the resulting solid was washed three times with 20 mL of methanol each time. Finally, the resulting solid was placed in a fume hood and allowed to air dry naturally to obtain the mitochondrial-targeting fluorescent dye of this invention, the structure of which is shown in formula (I): Equation (1) The mitochondrial-targeting fluorescent dye of this invention 1 H NMR spectrum as shown Figure 1 As shown, 13 The C NMR spectrum is as follows Figure 2 As shown,1 HNMR spectrum and 13 The C NMR spectrum corresponds to the chemical structure of the fluorescent dye, indicating that it was successfully synthesized.

[0025] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.33 (d, J = 13.4 Hz, 1H), 8.03 (d, J =7.5 Hz, 1H), 7.90 (d, J = 7.9 Hz, 1H), 7.62-7.43 (m,2H), 7.43-7.30 (m, 2H),7.24 (d, J = 7.8 Hz, 1H), 7.19-7.05 (m, 1H), 5.88 (d, J = 13.4 Hz, 1H), 3.46(s, 3H), 1.65 (s, 6H).

[0026] 13 C NMR (126 MHz, DMSO)δ170.26, 165.17,154.14, 144.82, 143.65, 140.04,133.61, 128.72, 127.07, 124.90, 123.46, 122.53, 122.38, 122.09, 117.70,110.00, 93.84, 91.43, 48.04, 30.48, 28.77.

[0027] Example 2: Evaluation of Mitochondrial Targeting HeLa cells were cultured in 25mm glass-bottomed culture dishes. The mitochondrial-targeting fluorescent dye synthesized in Example 1 (5 μM) was added to the cell culture medium, and the cells were incubated at 37°C for 10 minutes. Subsequently, the commercially available mitochondrial fluorescent dye Mito-red (5 μM) was added to the cell culture medium, and incubation continued for another 10 minutes. Finally, the cells were washed twice with PBS and fresh culture medium was added. The fluorescence imaging of HeLa cells was observed using a confocal fluorescence microscope. The green channel (Ex = 488 nm, Em = 500-550 nm) of the mitochondrial-targeting fluorescent dye (Formula (I)) and the red channel (Ex = 543 nm, Em = 600-650 nm) of Mito-red were also observed.

[0028] like Figure 3 As shown, the green and red fluorescent regions within the cells highly overlap, with a correlation coefficient Rr of 0.96. These results indicate that the mitochondrial-targeting fluorescent dye of this invention exhibits excellent mitochondrial targeting.

[0029] Example 3 Evaluation of Cellular Uptake Rate HeLa cells were cultured in 25 mm glass-bottomed culture dishes. The mitochondrial-targeting fluorescent dye (5 μM) synthesized in Example 1 was added to the cell culture medium. Fluorescence images of HeLa cells taking up the fluorescent dye were captured using a confocal fluorescence microscope in time-scan mode (Ex = 488 nm, Em = 500-555 nm).

[0030] like Figure 4 As shown, after adding the mitochondrial-targeting fluorescent dye of the present invention for 10 seconds, a weak fluorescent signal appeared in HeLa cells. As the incubation time increased, the fluorescent signal in the cells gradually increased and reached a relatively stable level after 50 seconds.

[0031] The above results indicate that the mitochondrial-targeting fluorescent dye of the present invention has good cell membrane penetration and can be rapidly taken up by cells.

[0032] Example 4: Evaluation of photostability Following the method in Example 2, the mitochondrial-targeting fluorescent dye synthesized in Example 1 and Mito-red were co-incubated with HeLa cells, respectively.

[0033] like Figure 5 As shown, from left to right, the images depict the fluorescence of cells after 1, 10, 20, 30, 40, and 50 scans. The figures demonstrate that the mitochondrial-targeting fluorescent dye of this invention still emits bright fluorescence after 50 scans, while the fluorescence intensity of the Mito-red fluorescent dye rapidly decreases with increasing scan count, and completely quenches after 30 scans.

[0034] The above results confirm that the mitochondrial-targeting fluorescent dye of the present invention has good photostability, which is beneficial for long-term imaging monitoring of mitochondria.

[0035] Example 5 Cytotoxicity Evaluation HeLa cells were fed at a concentration of 1.5 × 10⁻⁶. 5 Cells were seeded at a density of 100 cells / well into 96-well microplates, and 100 μL of cell culture medium was added. The plates were incubated at 37°C for 24 hours. The culture medium was then aspirated, and 100 μL of cell culture medium containing different concentrations (0-50 μM) of the mitochondrial-targeting fluorescent dye synthesized in Example 1 was added. The plates were incubated for another 24 hours. The culture medium was then aspirated, and 100 μL of cell culture medium containing CCK-8 was added. After 2 hours of incubation, the absorbance at 450 nm was measured using a microplate reader.

[0036] like Figure 6As shown, after incubation with ≤50μM of the mitochondrial-targeting fluorescent dye of the present invention for 24 hours, HeLa cells still maintained a survival rate of over 83.8%, indicating that the mitochondrial-targeting fluorescent dye of the present invention has low cytotoxicity.

[0037] Example 6: Dynamic monitoring of mitochondrial morphological changes HeLa cells were cultured in 25 mm glass-bottomed culture dishes, and the mitochondrial-targeting fluorescent dye synthesized in Example 1 (5 μM) was added to the cell culture medium. The cells were incubated at 37 °C for 10 minutes. Subsequently, ellastatin (10 μM) was added to the cell culture medium to induce ferroptosis, and fluorescence images of the cells taking up the mitochondrial-targeting fluorescent dye were captured using a confocal fluorescence microscope in time-scan mode (Ex = 488 nm, Em = 500-550 nm).

[0038] like Figure 7 As shown, as the incubation time was gradually extended to 30 minutes, the volume of mitochondria in HeLa cells was clearly observed to decrease, indicating that the mitochondrial-targeting fluorescent dye of the present invention can be used to dynamically monitor changes in mitochondrial morphology.

[0039] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various equivalent transformations can be made to the technical solutions of the present invention, and these equivalent transformations all fall within the protection scope of the present invention.

Claims

1. A mitochondria-targeting fluorescent dye, characterized in that, The structural formula of the mitochondria-targeting fluorescent dye is shown as formula (I). Formula (I).

2. The method of synthesis of a mitochondria-targeting fluorescent dye according to claim 1, characterized in that, The method comprises the following steps: The benzothiazole-2-acetonitrile and the Fischer aldehyde are added into an organic solvent, and heated to react under the action of a base to obtain the fluorescent dye shown as formula (I).

3. The method of synthesis of claim 2, wherein, The molar ratio of the benzothiazole-2-acetonitrile to the Fischer aldehyde is 1: (1-4).

4. The method of synthesis of claim 2, wherein, The organic solvent is at least one of methanol, ethanol, acetonitrile, acetone, 1, 4-dioxane, tetrahydrofuran or N, N-dimethylformamide.

5. The method of synthesis of claim 2, wherein, The base is at least one of cesium carbonate, potassium carbonate, sodium carbonate, sodium bicarbonate, pyridine, piperazine, triethylamine or N, N-dimethylaminopyridine.

6. The method of synthesis of claim 2, wherein, The molar ratio of the benzothiazole-2-acetonitrile to the base is 1: (1-4).

7. The method of synthesis of claim 2, wherein, The heating temperature is 20-60 DEG C, and the reaction time is 4-12 hours.

8. The mitochondria-targeting fluorescent dye of claim 1 is used as a mitochondrial fluorescent probe.

9. The mitochondria-targeting fluorescent dye of claim 1 is used in intracellular mitochondrial fluorescent imaging and morphological monitoring for the purpose of non-disease diagnosis and treatment.

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