Rhodamine fluorescent probe for mitochondrial dynamics sted imaging and preparation method thereof

Abstract

The application discloses a kind of rhodamine fluorescent probe of mitochondrial dynamic STED imaging and preparation method thereof, method includes: compound A is mixed with compound B and added to initial solvent, stirring and heating reaction 20-50 hours, after reaction solution is cooled to room temperature, neutralizing agent is added to neutralize;Using organic solvent, the solution obtained after neutralization is extracted, organic layer is collected and dried by Na2SO4, then solvent is evaporated using rotary evaporator;The product obtained after evaporation is dissolved in reaction solvent and compound C is added to react 8-24 hours, under the condition of reduced pressure, remove solvent by distillation, the residue obtained is purified by column chromatography, and the fluorescent probe in solid form is obtained.The fluorescent probe prepared by the above method is a kind of small organic molecule based on rhodamine framework with high brightness, high erasing efficiency, anti-blinking and low toxicity, which can realize the dynamic imaging of live cell mitochondria ridge and achieve 84nm ultra-high resolution, thereby greatly improving the imaging effect.

Description

Technical Field

[0001] This invention belongs to the field of compound synthesis technology, and particularly relates to a rhodamine fluorescent probe for mitochondrial dynamic STED imaging and its preparation method. Background Technology

[0002] Currently, various super-resolution microscopy techniques are used to study the submicroscopic structure of mitochondria in cells. Stochastic optical reconstruction microscopy (STORM) has revealed the process of cristae formation, with cristae in COS7 and U2OS cells primarily appearing as linear and punctate structures, respectively. Although STORM can achieve a resolution of approximately 22 nm, real-time dynamic imaging of mitochondrial cristae remains challenging due to the time required for image reconstruction. Structured illumination microscopy (SIM) can achieve dynamic imaging of mitochondrial cristae, but artifacts occur during algorithmic reconstruction, making it difficult to guarantee fidelity and quantitative characteristics. In contrast, STED's high spatial and temporal resolution makes it the most feasible choice for studying mitochondria.

[0003] STED microscopy can surpass the Abbe diffraction limit and visualize structural features within mitochondria. However, obtaining live cells with STED over extended periods remains a challenge. First, most fluorophores have limited photostability, their fluorescence intensity deactivating within seconds. Fluorophore stability is a particular concern in STED microscopy, as samples exposed to exceptionally powerful depletion lasers can achieve higher resolutions. Second, existing dyes for labeling mitochondria have practical drawbacks: their photostability is insufficient for high-resolution, long-term STED imaging, and most of these dyes have excitation wavelengths above 600 nm, making dyes in the 500-600 nm range difficult to use. Therefore, fluorescent probes in existing techniques suffer from poor imaging performance. Summary of the Invention

[0004] This invention provides a rhodamine fluorescent probe for mitochondrial dynamic STED imaging and its preparation method, aiming to solve the problem of poor imaging effect of fluorescent probes in the prior art.

[0005] In a first aspect, embodiments of the present invention provide a method for preparing a rhodamine fluorescent probe for dynamic STED imaging of mitochondria, wherein the method includes:

[0006] Compound A and compound B are mixed and added to the initial solvent, and the mixture is stirred and heated to react for 20-50 hours; wherein compound A is a mixture of 4-(1,2,2-triphenylvinyl)benzaldehyde and p-benzenesulfonic acid monohydrate, and compound B is 3-diethylaminophenol;

[0007] After the reaction solution has cooled to room temperature, a neutralizing agent is added for neutralization.

[0008] The solution obtained after neutralization was extracted with an organic solvent, the organic layer was collected and dried with Na2SO4, and then the solvent was evaporated using a rotary evaporator.

[0009] The product obtained after evaporation was dissolved in a reaction solvent and compound C was added. After reacting for 8-24 hours, the solvent was removed by distillation under reduced pressure. The residue was purified by column chromatography to obtain a solid fluorescent probe. The compound C was concentrated H2SO4 or tetrahydrotetrachlorobenzoquinone.

[0010] The method for preparing a rhodamine fluorescent probe for mitochondrial dynamic STED imaging includes the following: compound A is a mixture of 4-(1,2,2-triphenylvinyl)benzaldehyde and p-benzenesulfonic acid monohydrate; compound B is 3-diethylaminophenol; the initial solvent is propionic acid; the reaction temperature is 65-75℃; the neutralizing agent is an aqueous solution of sodium acetate; and the organic solvent is chloroform.

[0011] The method for preparing the rhodamine fluorescent probe for mitochondrial dynamic STED imaging, wherein the molar ratio of 4-(1,2,2-triphenylvinyl)benzaldehyde to p-benzenesulfonic acid monohydrate in compound A is (10-50):1, and the molar ratio of compound B to compound A is (0.6-2.5):1.

[0012] The method for preparing rhodamine fluorescent probes for mitochondrial dynamic STED imaging, wherein the step of dissolving the product obtained after evaporation in a reaction solvent and adding compound C to react for 8-24 hours includes:

[0013] The product obtained after evaporation is dissolved in a reaction solvent, and tetrahydrotetrachlorobenzoquinone is slowly added; wherein the reaction solvent is chloroform, and the volume ratio of the reaction solvent to the initial solvent is (1.5-2.5):1;

[0014] Stir the reaction mixture at room temperature for 8-10 hours.

[0015] The method for preparing the rhodamine fluorescent probe for mitochondrial dynamic STED imaging, wherein the eluent in the column chromatography purification is a mixed solution of dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol is (26-34):1.

[0016] Secondly, embodiments of the present invention also provide a rhodamine fluorescent probe for mitochondrial dynamic STED imaging, which is prepared using the preparation method described in the first aspect above.

[0017] This invention provides a rhodamine fluorescent probe for dynamic STED imaging of mitochondria and its preparation method. The method includes: mixing compound A and compound B and adding them to an initial solvent; stirring and heating the mixture for 20-50 hours; after the reaction solution cools to room temperature, adding a neutralizing agent for neutralization; extracting the neutralized solution with an organic solvent, collecting the organic layer and drying it with Na2SO4, then evaporating the solvent using a rotary evaporator; dissolving the evaporated product in the reaction solvent and adding compound C for 8-24 hours; removing the solvent by distillation under reduced pressure; purifying the residue by column chromatography to obtain a solid fluorescent probe. The fluorescent probe prepared by the above method is a small organic molecule based on the rhodamine architecture with high brightness, high erasure efficiency, bleach resistance, and low toxicity. It can achieve dynamic imaging of the mitochondrial crest in live cells with an ultra-high resolution of 84 nm, thereby significantly improving the imaging effect. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a flowchart of a method for preparing a rhodamine fluorescent probe for dynamic STED imaging of mitochondria provided in an embodiment of the present invention;

[0020] Figure 2 A schematic diagram illustrating the application effect of the rhodamine fluorescent probe for mitochondrial dynamic STED imaging provided in this embodiment of the invention.

[0021] Figure 3 This is a schematic diagram illustrating another application effect of the rhodamine fluorescent probe used for imaging dynamic STED mitochondrial ... imaging provided in this embodiment of the invention.

[0022] Figure 4 This is a schematic diagram illustrating another application effect of the rhodamine fluorescent probe for imaging dynamic STED mitochondrial imaging provided in this embodiment of the invention.

[0023] Figure 5 This is a schematic diagram illustrating another application effect of the rhodamine fluorescent probe for imaging dynamic STED mitochondrial imaging provided in this embodiment of the invention. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0026] It should also be understood that the terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0027] It should also be further understood that the term "and / or" as used in this specification and the appended claims refers to any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0028] In this embodiment, please refer to Figure 1 , Figure 1 This is a flowchart illustrating the method for preparing a rhodamine fluorescent probe for dynamic STED imaging of mitochondria according to an embodiment of the present invention. As shown in the figure, this embodiment of the present invention provides a method for preparing a rhodamine fluorescent probe for dynamic STED imaging of mitochondria, including steps S110 to S140.

[0029] S110. Mix compound A and compound B and add them to the initial solvent. Stir and heat to react for 20-50 hours.

[0030] Compound A is a mixture of 4-(1,2,2-triphenylvinyl)benzaldehyde and p-benzenesulfonic acid monohydrate, and compound B is 3-diethylaminophenol. The initial solvent is propionic acid, and the reaction requires heating to 65-75°C. The corresponding reaction formula for this process is: .

[0031] In compound A, the molar ratio of 4-(1,2,2-triphenylvinyl)benzaldehyde to p-benzenesulfonic acid monohydrate is (10-50):1, and the molar ratio of compound B to compound A is (0.6-2.5):1.

[0032] S120. After the reaction solution has cooled to room temperature, add a neutralizing agent to neutralize it.

[0033] After the reaction solution cools to room temperature, a neutralizing agent is added to neutralize it, thus bringing the pH of the neutralized solution to between 6 and 8. Compound A is a mixture of 4-(1,2,2-triphenylvinyl)benzaldehyde and p-benzenesulfonic acid monohydrate, compound B is 3-diethylaminophenol, and the neutralizing agent is an aqueous solution of sodium acetate.

[0034] S130. Extract the neutralized solution with an organic solvent, collect the organic layer and dry it with Na2SO4, and then evaporate the solvent using a rotary evaporator.

[0035] The neutralized solution is extracted using an organic solvent, which can be performed 2-4 times. The organic layer is collected by extraction and dried with anhydrous Na2SO4. The solvent is then removed by distillation under reduced pressure using a rotary evaporator.

[0036] Compound A is a mixture of 4-(1,2,2-triphenylvinyl)benzaldehyde and p-benzenesulfonic acid monohydrate, and compound B is 3-diethylaminophenol. The organic solvent is chloroform. The specific operating steps are as follows: the product obtained after evaporation is dissolved in the reaction solvent, and tetrahydrotetrachlorobenzoquinone is slowly added; wherein the reaction solvent is chloroform, and the volume ratio of the reaction solvent to the initial solvent is (1.5-2.5):1; the reaction mixture is stirred at room temperature for 8-10 hours.

[0037] S140. After dissolving the product obtained after evaporation in the reaction solvent and adding compound C, the reaction proceeds for 8-24 hours. The solvent is then removed by distillation under reduced pressure. The residue is purified by column chromatography to obtain a solid fluorescent probe.

[0038] The residual product obtained after rotary evaporation was dissolved in the reaction solvent and compound C was added to carry out the reaction.

[0039] If compound A is 4-(1,2,2-triphenylvinyl)phenol, then methanol solution is used as the reaction solvent, and concentrated H2SO4 is used as compound C; the eluent in column chromatography purification is a mixed solution of dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol is (45-60):1.

[0040] If compound A is a mixture of 4-(1,2,2-triphenylvinyl)benzaldehyde and p-benzenesulfonic acid monohydrate, then chloroform is used as the reaction solvent. Compound C is tetrahydrotetrachlorobenzoquinone. The eluent in column chromatography purification is also a mixed solution of dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol is (26-34):1.

[0041] This invention also provides a rhodamine fluorescent probe for mitochondrial dynamic STED imaging, which is prepared using the method described in the above embodiments. The specific preparation process of the fluorescent probe is illustrated in the following examples.

[0042] Example 1

[0043] Compound A is a mixture of 4-(1,2,2-triphenylvinyl)benzaldehyde and p-benzenesulfonic acid monohydrate, and compound B is 3-diethylaminophenol. The amounts of 4-(1,2,2-triphenylvinyl)benzaldehyde in the reaction are 1 mmol–3 mmol, p-benzenesulfonic acid monohydrate is 0.05 mmol–0.1 mmol, and 3-diethylaminophenol is 2 mmol. The initial solvent is propionic acid, with a volume of 10–20 mL. After placing compounds A and B in the initial solvent, the mixture was stirred and heated at 70 °C for 24 hours to allow for complete reaction. After cooling to room temperature, the mixture was neutralized with an aqueous sodium acetate solution (2 mol / L). The resulting suspension was extracted with chloroform as the organic solvent, and the combined organic extracts were dried with anhydrous sodium sulfate. After evaporating the solvent using a rotary evaporator, the solid product was dissolved in approximately 30 mL of chloroform, and then tetrahydrotetrachlorobenzoquinone (2.4 mmol, 590 mg) was added. The mixture was stirred at room temperature for 8 hours and then concentrated by rotary evaporation. The residue was purified by silica gel column chromatography using the mixture as eluent in dichloromethane / methanol (v / v = 30 / 1) to give compound TPERh (0.23 g, 35.6%).

[0044] Imaging tests were performed on the fluorescent probe TPERh obtained in Example 1 above. For example, live SOV3 cells were mixed in DMEM containing the fluorescent probe TPERh (0.2 μL, 1 mM) and 10% fetal bovine serum and stained in a CO2 incubator for 15 minutes. The cells were then washed three times with PBS to remove the free probe and incubated with fresh culture medium for 30 minutes. Then, a mitochondrial tracker (MitoTracker@Deep Red FM 644 / 665, 0.5 μL, 1 mM) or a lysosomal tracker (FluoLyso Deep Red 649 / 665, 0.5 μL, 1 mM) was added and the cells were incubated in a culture dish for 20 minutes. After removing the waste liquid, the cells were washed three times with PBS, added with fresh culture medium, and incubated in an incubator for 30 minutes. Confocal co-localization imaging was then performed, and the resulting images are shown below. Figure 2 As shown. Figure 2In Figure 2, MI-tracker represents one type of fluorescence signal in the red channel, ER-tracker represents another type of fluorescence signal in the red channel, Rh-based dye represents the image obtained by staining with TPERh alone, Merge represents the image obtained by merging the fluorescence signals in the red and green channels, and R represents the correlation coefficient between the two channels. As shown in Figure 2, the fluorescence signal of TPERh (green channel) is perfectly co-localized with the fluorescence signal of MI-tracker (red channel), with a Pearson correlation coefficient of 0.920, while the co-localization with the fluorescence signal of ER-tracker (red channel, Pearson correlation coefficient) is poor (lysosome co-localization coefficient is 0.295). The high Pearson correlation coefficient between TPERh and mitochondria clearly indicates that the TPERh probe can specifically target mitochondria in living cells.

[0045] Furthermore, STED imaging was performed using a Leica TCS SP8 STED system with excitation and depletion wavelengths of 560 nm and 660 nm, respectively. A HyD detector and a STED WHITE objective lens (80× / 1.40 oil) were used. Unless otherwise noted, STED images were acquired with 560 nm (1 μW) excitation, a 570–600 nm detector detection range, and a 660 nm depletion laser (CW-STED, 23 mW). To determine the minimum TPERh concentration for STED imaging, we conducted STED imaging experiments at different concentrations (excitation wavelength: 540 nm, erase wavelength: 660 nm, excitation power: 1 µW, erase power: 23 mW), with TPERh probe concentrations of 0.2 µM, 0.4 µM, 0.5 µM, 1.2 µM, 1.4 µM, and 1.6 µM, respectively. Images were processed using Lecia X, and the full width at half maximum (FWHM) resolution was determined to be 84 nm based on Gaussian fitting of the mitochondrial signal intensity distribution. The resulting imaging images are shown below. Figure 3 As shown, Figure 3 The image shows images of mitochondrial cristae obtained with different concentrations of the TPERh probe; subsequent dynamic imaging of the mitochondria clearly shows the fragmentation of the mitochondrial cristae, as shown in the dynamic imaging results. Figure 4 As shown. The raw data from the confocal and STED images clearly show the mitochondrial cristae, and the resulting images are shown in Figure 4(A) and 4(B), respectively. Figure 4 As shown in (B), the Raw image has no background removed; deconvolution is performed on the confocal and STED images to remove the background, and the resulting images are shown in Figure 4(C) and 4(B), respectively. Figure 4As shown in (D), Decon is used to remove the background. Compared with the mitochondrial inner membrane confocal image with a resolution of 330 nm, the STED imaging results revealed the fine structure of the cristae inside the mitochondria at a resolution of 84 nm. The obtained images are shown in Figure 4(F), where frame represents the frame number. Utilizing the high resolution of the TPERh probe in mitochondrial fluorescence imaging, STED can be used to monitor dynamic processes and subtle morphological changes within mitochondria (excitation wavelength: 540 nm, erase wavelength: 660 nm, excitation power: 1 µW, erase power: 23 mW, probe staining concentration: 0.1 µM). The signal intensity analysis results obtained by analyzing the confocal image and the STED image are as follows: Figure 5 As shown, the TPERh fluorescent probe has a strong detection depth position based on a resolution of 84 nm, so a resolution of 84 nm is chosen for fluorescence signal detection.

[0046] This invention discloses a rhodamine fluorescent probe for dynamic STED imaging of mitochondria and its preparation method. The method includes: mixing compound A and compound B and adding them to an initial solvent, stirring and heating the mixture for 20-50 hours; after the reaction solution cools to room temperature, adding a neutralizing agent for neutralization; extracting the neutralized solution with an organic solvent, collecting the organic layer and drying it with Na2SO4, then evaporating the solvent using a rotary evaporator; dissolving the evaporated product in the reaction solvent and adding compound C to react for 8-24 hours; removing the solvent by distillation under reduced pressure; purifying the residue by column chromatography to obtain a solid fluorescent probe. The fluorescent probe prepared by the above method is a small organic molecule based on the rhodamine architecture with high brightness, high erasure efficiency, bleach resistance, and low toxicity. It can achieve dynamic imaging of the mitochondrial crest in live cells with an ultra-high resolution of 84 nm, thereby significantly improving the imaging effect.

[0047] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing a rhodamine fluorescent probe for dynamic STED imaging of mitochondria, characterized in that, The methods include: Compound A and compound B are mixed and added to the initial solvent, and the mixture is stirred and heated to react for 20-50 hours; wherein compound A is a mixture of 4-(1,2,2-triphenylvinyl)benzaldehyde and p-benzenesulfonic acid monohydrate, and compound B is 3-diethylaminophenol; After the reaction solution has cooled to room temperature, a neutralizing agent is added for neutralization. The solution obtained after neutralization was extracted with an organic solvent, the organic layer was collected and dried with Na2SO4, and then the solvent was evaporated using a rotary evaporator. The product obtained after evaporation was dissolved in a reaction solvent and compound C was added. After reacting for 8-24 hours, the solvent was removed by distillation under reduced pressure. The residue was purified by column chromatography to obtain a solid fluorescent probe. The compound C is tetrahydrotetrachlorobenzoquinone.

2. The method for preparing a rhodamine fluorescent probe for mitochondrial dynamic STED imaging according to claim 1, characterized in that, Compound A is a mixture of 4-(1,2,2-triphenylvinyl)benzaldehyde and p-benzenesulfonic acid monohydrate, compound B is 3-diethylaminophenol, the initial solvent is propionic acid, the reaction temperature is 65-75℃, the neutralizing agent is sodium acetate aqueous solution, and the organic solvent is chloroform.

3. The method for preparing rhodamine fluorescent probes for mitochondrial dynamic STED imaging according to claim 2, characterized in that, The molar ratio of 4-(1,2,2-triphenylvinyl)benzaldehyde to p-benzenesulfonic acid monohydrate in compound A is (10-50):1, and the molar ratio of compound B to compound A is (0.6-2.5):

1.

4. The method for preparing a rhodamine fluorescent probe for mitochondrial dynamic STED imaging according to claim 2 or 3, characterized in that, The step of dissolving the product obtained after evaporation in a reaction solvent and adding compound C to react for 8-24 hours includes: The product obtained after evaporation is dissolved in a reaction solvent, and tetrahydrotetrachlorobenzoquinone is slowly added; wherein the reaction solvent is chloroform, and the volume ratio of the reaction solvent to the initial solvent is (1.5-2.5):1; Stir the reaction mixture at room temperature for 8-10 hours.

5. The method for preparing a rhodamine fluorescent probe for mitochondrial dynamic STED imaging according to claim 4, characterized in that, The eluent used in the column chromatography purification was a mixed solution of dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol was (26-34):1.

Images