Novel quinoline derivative probe as well as preparation method and application thereof

Abstract

The invention discloses a novel quinoline derivative probe (HD) as well as a preparation method and application thereof, and belongs to the technical field of fluorescent probes. The probe has two different spectral switching modes, and this single molecule / dual mode design effectively eliminates the obstacle to deliver the two different probes to the mitochondria, as well as the additional toxicity associated with multi-probe coupling. The fluorescence behavior of HD in mitochondria proves the special ability of HD in monitoring protein and nucleic acid dynamics in living cells. Mitochondrial protein and nucleic acid are fused to form nucleoprotein, and the nucleoprotein is dissociated and is in a balanced state under normal conditions. This balance regulates the activities necessary for the mitochondria, including fusion and division. The invention opens up a new thought for reasonable design of the mitochondrial nucleoprotein dynamic super-resolution imaging probe, and lays a foundation for further research on the action of interaction of mitochondrial nucleic acid and protein in the pathogenesis and treatment strategy of Parkinson's disease.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of fluorescent probe, and particularly relates to a novel quinoline derivative probe, a preparation method and application thereof. BACKGROUND

[0002] As an emerging fluorescence imaging technique, super-resolution imaging, especially stimulated emission depletion (STED) nanoscopy, provides sub-diffraction resolution while retaining the advantages of fluorescence confocal microscopy for live cell imaging. This technique has important potential applications in various biological fields, including visualizing the precise structure and dynamic processes of subcellular organelles in live cells at the nanoscale, and monitoring the spatial distribution of bioactive molecules (such as metal ions, reactive oxygen species (ROS), reactive nitrogen species (RNS), nucleic acids, and proteins) within subcellular organelles and their interactions in real time and in situ. For example, Peng et al. designed a pair of FRET fluorescent probes (OR-LA and SiR-BA) for visualizing mitochondrial membrane potential monitoring, enabling long-term super-resolution imaging of mitochondria. Zheng et al. developed a ratiometric fluorescent probe SRF-HClO, which was successfully applied to super-resolution imaging of lysosomal HClO in cancer cells during ferroptosis. Despite some progress, there are few reports on the use of STED to study the dynamic behavior of nucleic acids, proteins, and their complex nucleoproteins in mitochondria. This scarcity is mainly due to the following difficulties: 1) STED imaging requires probes with high resistance to photobleaching; 2) the lack of unique probes to distinguish nucleic acids and proteins in mitochondria; 3) the high cytotoxicity associated with the simultaneous use of nucleic acid and protein reactive probes can distort the observed results. Therefore, there is an urgent need to develop a high-quality probe suitable for STED nanoscopy that can simultaneously and highly selectively label mitochondrial nucleic acids and proteins. SUMMARY

[0003] The present application aims to provide a new quinoline derivative probe (HD) and its preparation method and application, which has two different spectral switch modes for rapid super-resolution imaging of mitochondrial proteins and nucleic acids. This single molecule / two mode design effectively eliminates the obstacles of delivering two different probes to mitochondria, and the additional toxicity related to multi-probe coupling. The fluorescence behavior of HD in mitochondria proves its special ability to monitor protein and nucleic acid dynamics in living cells. Subcellular organelle level STED imaging shows that mitochondrial proteins fuse with nucleic acids to form nucleoproteins, and the dissociation of nucleoproteins is in equilibrium under normal conditions. And this balance regulates the activities essential for mitochondria, including fusion and fission. An important finding is that the progression of Parkinson's disease is related to the dissociation of nucleoproteins. Therefore, this work opens up new ideas for the rational design of mitochondrial nucleoprotein dynamics super-resolution imaging probes, and lays a foundation for further studying the role of mitochondrial nucleic acid and protein interaction in the pathogenesis and treatment strategies of Parkinson's disease.

[0004] The purpose of the present application can be achieved by the following technical solutions:

[0005] A new quinoline derivative probe has the following structure:

[0006]

[0007] The second purpose of the present application is to provide a preparation method of the new quinoline derivative probe, which is synthesized by reacting p-dimethylaminobenzaldehyde and compound 1.

[0008] The reaction equation is as follows:

[0009]

[0010] Further, the reaction molar ratio of p-dimethylaminobenzaldehyde and compound 1 in the above reaction process is 1:1.

[0011] Further, the p-dimethylaminobenzaldehyde and compound 1 in the above reaction process are reacted at 83 DEG C.

[0012] Further, the p-dimethylaminobenzaldehyde and compound 1 in the above reaction process are reacted under ethanol refluxing condition, and piperidine and glacial acetic acid are used as catalysts.

[0013] Further, after the reaction, the solvent is removed by rotary evaporation under vacuum, and purified by silica gel chromatography, eluted with CH2Cl2:CH3OH=50:1 (V / V).

[0014] The third object of the present application is to provide the novel quinoline derivative probe with two different spectral switch modes for rapid super-resolution imaging of mitochondrial proteins and nucleic acids, for tracking the dynamic changes of mitochondrial nucleoproteins, and particularly for use in a Parkinson's disease model.

[0015] The excitation wavelength of the probe of the present application is 555 nm, and the maximum emission wavelength is 655 nm. The molecule forms a twisted intramolecular charge transfer structure through a carbon-carbon double bond, and N,N-dimethylaniline serves as a molecular rotor, so that the synthesized probe has no fluorescence. When 2 mg / ml BSA is added, the fluorescence intensity of HD increases by 44 times. In contrast, in the presence of nucleic acids, strong deep red fluorescence is emitted, and when the concentration of DNA or RNA is increased to 100 uM, the fluorescence intensity of HD increases by 30 times and 22 times, respectively. The emission wavelength when HD interacts with BSA is 60 nm different from the emission wavelength when it interacts with nucleic acids. This difference indicates that a single probe can simultaneously distinguish BSA and nucleic acids. HD has almost no fluorescence in PBS buffer, which demonstrates the selectivity of HD.

[0016] Advantages of the present application:

[0017] (1) The present application selects benzene N-dimethylaniline triphenyl and quinoline derivatives as the parent, so that the probe has good enrichment ability and significant transmembrane ability, HD selectivity and discrimination, so that it can rapidly distinguish BSA and nucleic acids. (2) The present application solves the problems of time-consuming and laborious, expensive, solution concentration relationship, high-resolution imaging monitoring of proteins with surface hydrophobic pockets and nucleic acids, and dynamic changes in the prior art. The present application designs and synthesizes a mitochondrial targeting probe HD with two different fluorescence switch modes, and the synthesis method is simple. At the same time, the balance between protein and nucleic acid assembly and disassembly is closely related to normal mitochondrial physiological activity, and the progression of Parkinson's disease is related to nucleoprotein relay. BRIEF DESCRIPTION OF DRAWINGS

[0018] The present application will be further described below in conjunction with the drawings.

[0019] Figure 1 The present application is a fluorescence probe HD 1 H NMR spectrum;

[0020] Figure 2 The present application is a fluorescence probe HD 13 H NMR spectrum;

[0021] Figure 3 The present application is a fluorescence probe HD HRMS spectrum;

[0022] Figure 4 The present application is a fluorescence probe HD (10 μM) in PBS (λ ex= 547 nm), PBS + BSA (λ ex = 555 nm), PBS + DNA (λ ex = 600 nm) and PBS + RNA (λ ex = 600 nm); (B-D) Fluorescence spectra of probe HD (10 μM) after addition of BSA, RNA (20 bp) and DNA (20 bp), respectively. The inset was taken under 365 nm UV light excitation. (E) Selectivity of HD (10 μM) towards different analytes (λ ex = 555 nm and 600 nm): 1 Blank, 2 Na + , 3 K + , 4 Ca 2+ , 5 Mg 2+ , 6 H2O2, 7 HCIO, 8 H2S, 9 Cysteine, 10 Serine, 11 Lysine, 12 Glutathione, 13 BSA, 14 Lysozyme, 15 Hemoglobin, 16 Trypsin, 17 Pepsin, 18 RNA and 19 DNA. The concentration of the rest of the samples was 100 μM except samples 13-17 (1 mg / mL). Error bars represent standard deviation (± SD);

[0023] Figure 5 is the fluorescence spectrum of the fluorescent probe HD of the present application when BSA (0.2 mg / mL) and DNA (2 μM) were added simultaneously. The excitation wavelength was 555 nm and the collection was from 570 to 650 nm;

[0024] Figure 6 is the fluorescence spectrum of the fluorescent probe HD of the present application when BSA (1 mg / mL) and DNA (100 μM) were added simultaneously. The excitation wavelength was 600 nm and the collection was from 720 to 800 nm;

[0025] Figure 7 is the MTT plot of the fluorescent probe HQBT of the present application;

[0026] Figure 8 is the confocal imaging of SH-SY5Y cells. (A) SH-SY5Y cells were incubated with 10 μM HD for 10 min. (B) SH-SY5Y cells were first incubated with 100 μM DTT for 3 h, and then incubated with HD for 10 min. (C) SH-SY5Y cells were incubated with 10 μM HD for 10 min, fixed with paraformaldehyde, permeabilized with Triton X-100, and then incubated with 10 mg / mL nuclease for 2 h;

[0027] Figure 9is the multicolor confocal imaging of protein and nucleic acid in mitochondria. SH-SY5Y cells were incubated with 10 μM HD for 10 min, and then incubated with Mito Tracker Green (0.5 μM). (A) bright field image. (B, C) fluorescence images of protein channel and nucleic acid channel. (D) Mito-Tracker Green fluorescence image (λ ex = 488 nm, λ em = 500-520 nm). (E-H) (A), (B), (C) superimposed with (D) respectively;

[0028] Figure 10 is the multicolor confocal imaging of protein and nucleic acid in mitochondria. HeLa cells were incubated with 10 μM HD for 10 min, and then incubated with Mito Tracker Green (0.5 μM). (A, B) fluorescence images of protein channel and nucleic acid channel. (C) Mito-Tracker Green fluorescence image (λ ex = 488 nm, λ em = 500-520 nm). (D-F) (A)+(C), (B)+(C) and (A)+(B) superimposed channels;

[0029] Figure 11 is the multicolor confocal imaging of protein and nucleic acid in mitochondria. Hep G2 cells were incubated with 10 μM HD for 10 min, and then incubated with Mito Tracker Green (0.5 μM). (A, B) fluorescence images of protein channel and nucleic acid channel. (C) Mito-Tracker Green fluorescence image (λ ex = 488 nm, λ em = 500-520 nm). (D-F) (A)+(C), (B)+(C) and (A)+(B) superimposed channels;

[0030] Figure 12 is the colocalization of the fluorescent probe HD of the present application with ER Tracker, LD Tracker and Lyso Tracker in cells;

[0031] Figure 13 is (A) conventional confocal imaging of protein and nucleic acid in mitochondria. (B) STED super-resolution imaging of protein and nucleic acid in mitochondria. In (A) and (B), SH-SY5Y cells were incubated with HD for 10 min. (C, D) are the statistical fluorescence intensity of the corresponding images in (A) and (B);

[0032] Figure 14 is the colocalization of the fluorescent probe HD of the present application with Rhodamine 123 in cells;

[0033] Figure 15 (A) Time-dependent STED imaging of single mitochondria. Probe-loaded cells were incubated in medium containing CCCP for 20 min, then in medium without CCCP for 20 min. (B) is a plot of the overlap coefficient versus time in (A). Error bars indicate standard deviation (±SD);

[0034] Figure 16 (A) Fluorescence signals of proteins and nucleic acids after adding different doses of 6-OHDA. (B) is a plot of the overlap coefficient (proteins and nucleic acids) versus 6-OHDA dose in (A). (C) Fluorescence signals of proteins and nucleic acids after different doses of MPP + + (D) is a plot of the overlap coefficient versus MPP + dose in (C). Error bars indicate standard deviation (±SD). DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0036] Please refer to Figures 1-16 The embodiments of the present application provide three technical solutions: a novel quinoline derivative probe, a preparation method and an application in real-time and super-resolution monitoring of mitochondrial nucleic acid-protein complex (nucleoprotein) dynamics, specifically including the following embodiments:

[0037] Embodiment one

[0038] The present application is a novel quinoline derivative probe. By selecting benzene N-xylene aniline triphenyl and quinoline derivative as the parent, the probe has good enrichment ability and significant transmembrane ability, HD selectivity and discrimination, so that it can quickly distinguish BSA and nucleic acid. The excitation wavelength of the probe is 555 nm, and the maximum emission wavelength is 655 nm. The molecule forms a twisted intramolecular charge transfer structure through a carbon-carbon double bond, and N, N-xylene aniline serves as a molecular rotor, so that the synthesized probe HD has no fluorescence;

[0039] The structural formula of the quinoline derivative probe is as follows:

[0040]

[0041] Embodiment two

[0042] This embodiment describes in detail the synthesis steps of the novel quinoline derivative probe HD.

[0043] Step 1: Add benzyl cyanobromide (0.50 g, 3.52 mmol) and 4-methylquinoline (0.68 g, 3.52 mmol) to 50 mL of acetonitrile and reflux for 12 h. Filter the precipitates and wash with ethanol to obtain the intermediate (0.33 g, 81% yield). The intermediate can be used directly in the next reaction without purification.

[0044] Step 1: To an ethanol solution containing p-dimethylaminobenzaldehyde (0.37 g, 2.50 mmol) and an intermediate (0.84 g, 2.50 mmol), piperidine (2 drops) and glacial acetic acid (2 drops) were added. The mixture was stirred and refluxed at 83 °C for 6 h. The solvent was removed by rotary evaporation under vacuum. The solution was purified by silica gel chromatography with a CH2Cl2:CH3OH ratio of 50:1 (V / V) to give HD as a black solid (0.96 g, 82% yield). The 1H NMR data are shown below. Figure 1 As shown, 1 ¹H NMR (400MHz, d⁶-DMSO) δ 9.34 (d, J = 6.7Hz, 1H), 9.07 (d, J = 8.8Hz, 1H), 8.48 (d, J = 6.8Hz, 1H), 8.31 (d, J = 15.5Hz, 1H), 8.17 (d, J = 8.8Hz, 1H), 8.11–8.03 (m, 2H), 7.89 (dd, J = 22.5, 8.6Hz, 5H), 7.50 (d, J = 8.4Hz, 2H), 6.85 (d, J = 8.8Hz, 2H), 6.28 (s, 2H), 3.10 (s, 6H). Carbon NMR data are as follows: Figure 2 As shown, 13 CNMR (101MHz, d6-DMSO) δ 154.91, 153.17, 147.51, 146.66, 140.85, 138.53, 135.48, 133.46, 132.36, 129.03, 128.25, 127.45, 126.82, 123.66, 119.50, 118.91, 114.73, 113.61, 112.53, 111.70, 58.43. HR-MS (m / z, ESI) theoretical value C 27 H 24 N3 + [M]m / z = 39270.2419653. Mass spectrometry data as follows: Figure 3 As shown, the experimental value is m / z = 390.1963.

[0045] Example 3

[0046] The properties of the quinoline derivative probe prepared in Example 2 were characterized in this example.

[0047] I. See Figures 4-6 As shown in Figure 4 Under 555 nm excitation, the emission peak centered at 655 nm can be clearly detected, which is consistent with the spectrum observed in the solution containing only BSA. The fluorescence intensity of HD binding DNA is low under this excitation wavelength, and the fluorescence spectrum overlap coefficient of HD binding BSA and HD binding DNA in the wavelength range of 570-650 nm is only 2.12% ( Figure 5 ), indicating that the fluorescence in this wavelength range is almost completely from HD binding BSA. Similarly, under 600 nm excitation, HD exhibits an emission peak centered at 710 nm, which is consistent with the emission peak of HD in the solution containing only DNA. The fluorescence spectrum overlap coefficient of HD binding DNA and BSA in the wavelength range of 720-800 nm is low, at 7.60% ( Figure 6 ), indicating that the fluorescence generated by HD binding to BSA under 620 nm excitation can be ignored. Therefore, using a single probe HD can simultaneously image proteins and nucleic acids without spectral overlap.

[0048] II. See Figures 7-8 , 10 μM HD (the concentration used for imaging) treatment shows that the cell viability is comparable to the control group. Even at a concentration twice that of the imaging experiment, the cell viability remains above 90%, indicating that the cytotoxicity of HD can be ignored ( Figure 7 ). Subsequently, HD was used for two-color fluorescence imaging of living cells (protein channel at 550 nm excitation, 570-650 nm range acquisition; nucleic acid channel at 620 nm excitation, 720-800 nm range acquisition). Confocal microscope imaging shows that after incubation of SH-SY5Y cells with 10 M HD, strong fluorescence signals are emitted in both the protein and nucleic acid channels ( Figure 8 A). In order to verify that the fluorescence activation under 555 nm and 600 nm excitation is due to the binding of proteins and nucleic acids to HD, respectively, we treated the cells with dithiothreitol (DTT) and nuclease, respectively, which degrade proteins and nucleic acids, respectively. As shown in Figure 8 B, compared with the control group, the fluorescence in the protein channel of SH-SY5Y cells treated with DTT disappears, while the fluorescence in the nucleic acid channel remains relatively unchanged. Similarly, treatment of SH-SY5Y cells with nuclease leads to loss of fluorescence in the nucleic acid channel, while the fluorescence in the protein channel is not affected ( Figure 8 C). These results indicate that the fluorescence activated under 555 nm and 600 nm excitation is due to the binding of proteins and nucleic acids to HD, respectively.

[0049] III. See Figures 9-12 , multicolor colocalization experiments using the commercial mitochondrial dye (Mito-Tracker Green) showed that the fluorescence signals of the protein and nucleic acid channels have a very high overlap coefficient with Mito-Tracker Green (0.93 and 0.90, respectively) Figure 9 ). Similar results were observed in Hep G2 and HeLa cells Figures 10-11 . In contrast, HD images co-stained with ER Tracker Green, Lyso Tracker Green, and LD Tracker Green showed very low Pearson correlation coefficients, 0.05 and 0.03, 0.07 and 0.06, 0.04 and 0.03, respectively Figure 12 , further confirming the specificity of probe HD for mitochondrial protein and nucleic acid imaging.

[0050] IV. See Figure 13 , the panels show the distribution of mitochondrial proteins and nucleic acids observed by confocal and STED microscopy. Using standard confocal microscopy, whose imaging resolution is not sufficient to achieve the subcellular organelle scale due to diffraction limitation, we observed that proteins with exposed hydrophobic pockets are distributed throughout the mitochondria, while nucleic acids are distributed in a slightly smaller area than proteins Figure 13 A). In contrast, high-resolution images obtained from STED microscopy show that these proteins are mainly located in the mitochondrial crista Figure 13 B). To demonstrate this, we performed a colocalization experiment using rhodamine 123, a commonly used dye for the inner mitochondrial membrane. The STED images show a major overlap between rhodamine 123 and the protein channel, indicating that these proteins are distributed on the inner mitochondrial membrane Figure 14 ). Fluorescence quantification figures further illustrate the high resolution and HD contrast of the STED microscopy images Figure 13 C, D). In addition, the distance between adjacent cristae is approximately 240-270 nm Figure 13 D), which is consistent with previous reports. Most notably, the super-resolution images show an overlap coefficient of 0.65 between mitochondrial proteins and nucleic acids Figure 13 B), indicating the formation of protein-nucleic acid complexes called nucleoproteins, which can be formed by the binding of proteins and nucleic acids, or can dissociate into individual proteins and nucleic acids according to the needs of the cell. These results suggest that probe HD provides an opportunity to study the dynamic physiological processes of mitochondrial proteins, nucleic acids, and nucleoproteins using STED microscopy.

[0051] V. See Figure 15, which shows the complete mitochondrial fusion process, the fluorescence intensity of protein and nucleic acid channels did not change significantly before and after fusion. Interestingly, the overlap coefficients of protein and nucleic acid channels maintained at about 0.76 before fusion. However, as the fusion began, these coefficients began to decrease, reaching a low of 0.43. Subsequently, as the fusion continued, the overlap coefficients of protein and nucleic acid gradually recovered to the level before fusion Figure 15 B). Similar phenomena were also observed during mitochondrial fission, as the mitochondrial fission proceeded, the overlap coefficients of protein and nucleic acid decreased from 0.78 to 0.41, and then gradually increased to 0.76. These results directly confirmed that the fusion and fission processes of mitochondria are closely related to the dynamic association and dissociation of proteins and nucleic acids.

[0052] VI. Refer to Figure 16 As shown in Figure 16 A, as the concentration of 6-OHDA gradually increased, the fluorescence intensity of protein and nucleic acid channels in mitochondria did not change significantly. In contrast, the overlap coefficients of protein and nucleic acid channels decreased sharply (from 0.8 to 0.5), indicating that the progression of Parkinson's disease is associated with a large dissociation of nucleoprotein complexes. In MPP + Similar phenomena were also observed in SH-SY5Y cells induced by MPP Figure 16 B). These findings are crucial for understanding the mechanisms of Parkinson's disease occurrence and development, and may provide new insights for its diagnosis and treatment.

[0053] In summary, we designed and synthesized a mitochondrial-targeting probe HD, which has two different fluorescence switch modes, allowing us to simultaneously monitor the dynamics of proteins with surface hydrophobic pockets and nucleic acids by super-resolution imaging. Using STED imaging, we studied the distribution of mitochondrial proteins and nucleic acids, revealing that proteins mainly fuse with nucleic acids to form nucleoprotein. The balance between the assembly and disassembly of proteins and nucleic acids is closely related to normal mitochondrial physiological activities, such as fusion and fission. In addition, an important finding is that the progression of Parkinson's disease is associated with the dissociation of nucleoprotein. In summary, this imaging probe has the potential to become a valuable tool for studying the dynamics of Parkinson's disease-related mitochondrial nucleoprotein, and may inspire the design of other functional sensors for intracellular organelles and biomolecules.

[0054] The above specific embodiments section specifically introduces the analysis method involved in the present application. It should be noted that the above introduction is only to help those skilled in the art better understand the method and ideas of the present application, and is not a limitation on the related content. Those skilled in the art can also make appropriate adjustments or modifications to the present application without departing from the principles of the present application, and the above adjustments and modifications should also belong to the protection scope of the present application.

Claims

1. A novel quinoline derivative probe, characterized in that, The structure is as follows:

2. The method for preparing a novel quinoline derivative probe according to claim 1, characterized in that, The quinoline derivative probe is synthesized by reacting p-dimethylaminobenzaldehyde and compound 1; The reaction equation is as follows:

3. The method for preparing a novel quinoline derivative probe according to claim 2, characterized in that, The reaction molar ratio of the p-dimethylaminobenzaldehyde and compound 1 in the reaction process is 1:

1.

4. The method for preparing a novel quinoline derivative probe according to claim 2, characterized in that, The p-dimethylaminobenzaldehyde and compound 1 in the reaction process are reacted at 83°C.

5. The method for preparing a novel quinoline derivative probe according to claim 2, characterized in that, The p-dimethylaminobenzaldehyde and compound 1 in the reaction process are reacted under ethanol refluxing condition, and piperidine and glacial acetic acid are added as catalysts.

6. The method for preparing a novel quinoline derivative probe according to claim 2, characterized in that, After the reaction, the solvent is removed by rotary evaporation under vacuum, and purified by silica gel chromatography, eluted with CH2Cl2:CH3OH = 50:

1.

7. Application of the novel quinoline derivative probe according to claim 1 in rapid super-resolution imaging of mitochondrial proteins and nucleic acids.

8. Use according to claim 7, characterized in that, Application in a Parkinson's disease model.

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