A mitochondria-targeting water-soluble aggregation-induced emission dye and a preparation method thereof

Abstract

The present application relates to the field of luminescent material, in particular to an aggregation-induced emission fluorescent dye with targeting function and a preparation method thereof. The dye provided in the present application has good biocompatibility and complete water solubility, and can be applied to superfast no-wash low phototoxicity imaging of living cells, greatly helping the biological research related to living cells.

Description

Technical Field

[0001] This invention relates to the field of luminescent materials, specifically to an aggregation-induced emission fluorescent dye with targeting function and its preparation method. Background Technology

[0002] Aggregation-caused quenching (ACQ) refers to the phenomenon where the fluorescence of traditional organic light-emitting materials weakens or even stops when they are at high concentrations or in a solid state; that is, fluorescence quenching occurs when the material is in an aggregated state. In practical applications, this effect can have some negative consequences. For example, when hydrophobic organic light-emitting materials are added to water, a commonly used detection medium, the low solubility causes the luminescent molecules to aggregate in the water, leading to a decrease in luminescence intensity.

[0003] In 2001, Academician Tang Benzhong first proposed the concept of aggregation-induced emission (AIE), a highly efficient emission process in an aggregated state, which has attracted widespread attention from peers both domestically and internationally. Currently, hundreds of research groups in dozens of countries have conducted related research, covering multiple directions including new AIE molecules, AIE mechanisms, and AIE materials. Significant results have also been achieved in many fields such as optoelectronic devices, biological probes and imaging, chemical sensing, and smart materials applications. AIE has become a research hotspot in luminescent materials and photophysics, and was listed as the second of the top 10 research frontiers in chemistry in the "2015 Research Frontiers" report jointly released by the Documentation and Information Center of the Chinese Academy of Sciences and Thomson Reuters. Furthermore, in 2016, the *Nature* News Feature column featured AIE materials under the title "The nanolight revolution is coming," highlighting that AIE materials provide solutions to the problems existing in currently used quantum dots and luminescent polymer dots, representing a new generation of nanoluminescent materials. Therefore, developing water-soluble AI E molecules with long-wavelength emission, aggregation-induced emission properties, and low phototoxicity is of great significance, but the road ahead remains extremely challenging.

[0004] In the field of bio-optical imaging, fluorescence bio-imaging, as a powerful non-invasive imaging technique, has demonstrated unique advantages, especially the development and application of super-resolution fluorescence microscopy. Compared to traditional optical imaging, super-resolution fluorescence imaging is based on a larger-scale photon statistical data, fundamentally breaking the limitation of the original optical far-field diffraction limit on the ultimate resolution of optical systems, surpassing the optical resolution limit, and achieving nanometer-level resolution.

[0005] To further improve imaging resolution and quality, researchers aim to design fluorescent dyes with higher brightness and better photostability. One major class of used fluorescent chromophores consists of small organic molecule fluorescent chromophore compounds, such as rhodamine dyes, which are now widely used in super-resolution imaging. These compounds are simple to synthesize, easy to modify, and have low toxicity, making them a focus of attention in the development and application of small organic molecule fluorescent probes.

[0006] However, traditional rhodamine dyes are all aggregation-induced quenching dyes, which have poor water solubility, weak biocompatibility, and complex synthetic routes. At the same time, their fluorescence brightness is affected by the nonradiative transition process of intramolecular torsional charge transfer after excitation, and their imaging potential has not been fully released, which is not conducive to further applications. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and solve the problem of poor water solubility of traditional rhodamine dyes mentioned in the background art.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides an aggregation-induced emission dye, the characteristic structure of which is as follows: Figure 1 As shown.

[0010] A second aspect of the present invention provides a method for preparing an aggregation-induced emission dye, comprising: S1, dissolving potassium carbonate, MG-B, and Br-Rho-2 in N,N-dimethylformamide, protecting with nitrogen gas, adding tetrakis(triphenylphosphine)palladium for reaction, and purifying; S2, dissolving the purified product in dichloromethane, adding tetrachloro-p-benzoquinone for reaction, and purifying to obtain the dye. The structural formula of Br-Rho-2 is as follows: Figure 2 As shown; the structural formula of the MG-B is as follows: Figure 3 As shown.

[0011] Preferably, in step S1, the molar ratio of MG-B to Br-Rho-2 is 12:10.

[0012] Preferably, in steps S1 and S2, the molar ratio of potassium carbonate, tetra(triphenylphosphine)palladium, and tetrachloro-p-benzoquinone is 1000:0.8:15.

[0013] Preferably, in step S1, the reaction time is 12-24 hours.

[0014] Preferably, in step S2, the reaction time is 30 minutes.

[0015] In some embodiments, the purification in step S1 includes: extracting, washing, drying, and performing column chromatography on the post-reaction mixture. Preferably, in the column chromatography in step S1, the adsorbent is silica gel, and the eluent is a mixture of ethyl acetate / hexane at a volume ratio of 1:20 to 1:10.

[0016] In some embodiments, step S2, the purification includes performing column chromatography. Preferably, in the column chromatography of step S2, the adsorbent is silica gel, and the eluent is a methanol / dichloromethane mixture with a volume ratio of 1:10 to 1:5.

[0017] A third aspect of the present invention provides an application of an aggregation-induced emission dye in live-cell imaging and an application of an aggregation-induced emission dye in mitochondrial targeted staining.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] (1) This invention is the first to develop and synthesize a water-soluble organic small molecule fluorescent dye MG-Rho-2 with mitochondrial targeting, which has aggregation-induced emission properties.

[0020] (2) The MG-Rho-2 molecule provided by this invention has good biocompatibility and complete water solubility, and can be applied to ultrafast wash-free low phototoxicity imaging of living cells, which greatly assists biological research related to living cells.

[0021] (3) The dye MG-Rho-2 provided by this invention exhibits a better signal-to-noise ratio compared to commercially available mitochondrial-targeting dyes.

[0022] (4) The present invention uses a one-pot method to prepare dye MG-Rho-2, which is simple to operate and conducive to laboratory and industrial applications. Attached Figure Description

[0023] Figure 1 The structural formula of the fluorescent probe material MG-Rho-2;

[0024] Figure 2 The structural formula is Br-Rho-2;

[0025] Figure 3 The structural formula for MG-B;

[0026] Figure 4 The synthesis route diagram for MG-Rho-2;

[0027] Figure 5 The hydrogen nuclear magnetic resonance spectrum of MG-Rho-2;

[0028] Figure 6 The image shows the carbon NMR spectrum of MG-Rho-2.

[0029] Figure 7 Here is the high-resolution mass spectrum of MG-Rho-2;

[0030] Figure 8 The image shows the optical properties of MG-Rho-2. Figure 8 A represents the photoluminescence spectrum of MG-Rho-2 in methanol. Figure 8 B represents the fluorescence spectra of MG-Rho-2 in tetrahydrofuran / water mixtures at different ratios. Figure 8 C represents the change in luminescence intensity of MG-Rho-2 with water content;

[0031] Figure 9 Image of HeLa cells using a laser scanning confocal microscope (CLSM). Figure 9 A shows the incubation pattern of 5 μM MG-Rho-2 (lex = 560 nm, lem = 570-750 nm). Figure 9 B is the incubation diagram of Mito Tracker Green (lex = 488nm, lem = 500-560nm). Figure 9 C is the overlay image. Figure 9 D is the fitting plot of colocation coefficients. Detailed Implementation

[0032] The technical solution of this patent will be further described in detail below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0033] Rhodamine dyes are among the most widely used dye platforms in the field of fluorescent probes. Fluorescent probes constructed based on this platform are typically rhodamine-based pyroxene lactam derivatives. These derivatives are non-fluorescent on their own; however, upon interaction with a guest, the pyroxene lactam structure opens, resulting in strong fluorescence emission and a significant color change. The Czarnik group first reported the use of these properties in 1997 to synthesize a "rhodamine-hydrazine" Cu... 2+ Since the advent of fluorescent probes, various fluorescent probes based on the rhodamine dye platform have been developed.

[0034] Conventional rhodamine fluorescent dyes readily form dimers, leading to intermolecular quenching and a decrease in fluorescence intensity. Furthermore, most biochemical reactions and biological assays use water as a medium, and rhodamine's high hydrophobicity makes it poorly soluble in water. These drawbacks limit the further application of rhodamine-based fluorescent dyes. Introducing sulfonic acid functional groups into traditional rhodamine dyes is the most common method to increase their water solubility and prevent fluorescence quenching, but the synthesis is relatively complex.

[0035] This invention successfully developed a small organic molecule water-soluble probe based on the rhodamine backbone, exhibiting aggregation-induced emission properties, hereinafter referred to as MG-Rho-2. Thanks to the two quaternary ammonium groups in the MG-Rho-2 molecule, it possesses complete water solubility and can be applied to ultrafast, wash-free, low-phototoxicity imaging of live cells, which will greatly contribute to biological research related to live cells.

[0036] Example 1: Preparation of MG-Rho-2 and its characterization by proton, carbon, and mass spectrometry.

[0037] (1) Preparation of the target compound

[0038] MG-Rho-2 synthesis route as follows Figure 4 As shown, the specific steps include:

[0039] A mixture consisting of 10 mmol potassium carbonate, 0.12 mmol MG-B, and 0.1 mmol Br-Rho-2 was dissolved in 3 mL of N,N-dimethylformamide. After nitrogen protection, 0.008 mmol tetrakis(triphenylphosphine)palladium was added, and the mixture was refluxed for 12 h.

[0040] The reaction mixture was extracted with dichloromethane, washed three times with saturated sodium chloride solution, and the organic phases were combined and dried over anhydrous sodium sulfate. The crude product was concentrated and purified by column chromatography, using silica gel as the adsorbent and a mixture of ethyl acetate / hexane at a volume ratio of 1:20 to 1:10 as the eluent.

[0041] The purified product was dissolved in dichloromethane, and 0.15 mmol of tetrachloro-p-benzoquinone was added. The mixture was stirred at room temperature for 30 min in a one-pot manner.

[0042] The crude product was purified by silica gel column chromatography, with a methanol / dichloromethane mixture in a volume ratio of 1:10 to 1:5 as the eluent, to finally obtain the target compound as a dark green solid.

[0043] (2) Hydrogen spectroscopy characterization

[0044] The target compound was characterized by proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 5 As shown, the specific data is as follows: 1H NMR(400MHz, MeOD-d4)δ(ppm) 1 H NMR (400MHz, MeOD) δ 8.16-8.02 (m, 4H), 7.65-7.42 (m, 10H), 7.16-7.04 (m, 6H), 7.06-6.89 (m, 2H), 3.36 (t, J = 18.4Hz, 24H).

[0045] (3) Carbon spectroscopy characterization

[0046] The target compound was characterized by carbon NMR spectroscopy, and the results are as follows: Figure 6 As shown, the specific data is as follows: 13 C NMR (100MHz, MeOD-d4) δ (ppm) 176.2, 157.7, 157.4, 157.1, 157.1, 144.1, 143.7, 141.2, 140.5, 139.7, 139. 2, 135.5, 135.4, 132.1, 131.4, 130.6, 127.4, 127.0, 127.0, 127.0, 127.0, 114.3, 113.5, 112.9, 96.3, 39.7.

[0047] (4) High-resolution mass spectrometry characterization

[0048] The target compound was characterized by high-resolution mass spectrometry (HRMS), and electrospray ionization (ESI) was selected as the ionization method. The results are as follows: Figure 7 As shown, the molecular formula is C 46 H 46 N4O 2+ The calculated molecular weight is 335.18303, and the experimental molecular weight is 335.18307.

[0049] The above proton, carbon, and mass spectrometry results all confirm that the target chemical is MG-Rho-2, indicating that the preparation method of this scheme is feasible.

[0050] Example 2: Characterization of aggregation-induced emission properties of MG-Rho-2

[0051] like Figure 8 As shown, the aggregation-induced emission properties of MG-Rho-2 were investigated. The results showed that MG-Rho-2 had an absorption peak at 550 nm and an emission peak at 600 nm in methanol. Figure 8B and 8C show the luminescence intensity of MG-Rho-2 in aqueous tetrahydrofuran solutions with different proportions. The fluorescence intensity is very weak when the water content is 100%, and increases with increasing tetrahydrofuran content. The fluorescence intensity is highest at a tetrahydrofuran content of 90%, increasing by 132 times, demonstrating that the MG-Rho-2 synthesized in this method possesses excellent aggregation-induced emission properties.

[0052] Example 3: Characterization of MG-Rho-2 mitochondrial targeting properties

[0053] like Figure 9 As shown, the mitochondrial targeting properties of MG-Rho-2 were investigated. Figure 9 In Figure A, the mitochondrial region of HeLa cells was specifically stained with MG-Rho-2, and the structure of the mitochondria was clearly observed, indicating that MG-Rho-2 can be localized to the mitochondria of living cells. Figure 9 B was stained with the mitochondrial green fluorescent probe Mito Tracker Green, and the staining results were overlaid with those of MG-Rho-2 to obtain... Figure 9 C, it can be observed that the two almost completely overlap. Furthermore, the Pearson correlation coefficient Rr was used to represent the degree of linear dependence between the two variables, quantifying the overlap of staining regions between MG-Rho-2 and Mito Tracker Green. The results showed that Rr = 0.94, indicating that MG-Rho-2 has a specific targeting effect on mitochondria.

[0054] This invention provides an aggregation-induced emission dye, MG-Rho-2, which exhibits excellent biocompatibility, complete water solubility, and mitochondrial targeting properties, enabling its application in ultrafast, wash-free, low-phototoxicity imaging of live cells. Furthermore, this invention provides an innovative and simple method for preparing MG-Rho-2, and the obtained product has been confirmed to have the correct structure after characterization by 1H NMR spectroscopy, 1C NMR spectroscopy, and high-resolution mass spectrometry.

[0055] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. An aggregation-induced emission dye, characterized in that, The characteristic structure of the dye is shown in the following formula: 。 2. A process for the preparation of a dye according to claim 1, characterized in that, The method comprises: S1, potassium carbonate, Br-Rho-2, MG-B is dissolved in N, N-dimethylformamide, after nitrogen protection, add four (triphenylphosphine) palladium reaction and purification; The structure of the Br-Rho-2 is shown in the following formula: ； The structure of the MG-B is shown in the following formula: ； S2, the product after purification is dissolved in dichloromethane, add four chloro-p-benzoquinone and react, after purification, the dye is obtained.

3. The method of claim 2, wherein, In the step S1, the molar ratio of the MG-B and the Br-Rho-2 is 12:

10.

4. The method of claim 3, wherein, In the steps S1 and S2, the molar ratio of the potassium carbonate, the four (triphenylphosphine) palladium and the four chloro-p-benzoquinone is 1000:0.8:

15.

5. The method of claim 4, wherein, In the step S1, the reaction time is 12-24h.

6. The method of claim 5, wherein, In the step S2, the reaction time is 30min.

7. The method according to any one of claims 2-6, characterized in that, In the step S1, the purification includes: after reaction, the mixture is extracted, washed, dried and column chromatography.

8. The method according to any one of claims 2-6, characterized in that, In the step S2, the purification includes: column chromatography.

9. Use of an aggregation-induced emission dye in imaging of live cells, characterized in that, The application of the aggregation-induced emission dye is the application for non-disease diagnosis and treatment purposes.

10. Use of an aggregation-induced emission dye for mitochondrial-targeted staining development, characterized in that, The application of the aggregation-induced emission dye is the application for non-disease diagnosis and treatment purposes.

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