Method for preparing fluorescent probe for detecting change in mitochondrial viscosity in living organism and method for detecting same

Abstract

This invention discloses a method for preparing and detecting a fluorescent probe for detecting changes in mitochondrial viscosity in organisms, belonging to the field of chemical analysis and detection technology. Triphenylbromoethylene and a five-membered heteroaromatic compound containing aldehyde and boric acid groups are dissolved and reacted with a catalyst; isopropanol is removed by cooling and vacuum evaporation, followed by water extraction, solvent removal, and column chromatography to obtain ND-SF-1; ND-SF-1 is mixed with 4-pyridineacetonitrile, anhydrous ethanol is added, piperidine is added dropwise, the mixture is heated, cooled, and refluxed, then cooled to room temperature, and the precipitated solid is washed to obtain ND-SF-2; ND-SF-2, 3-bromopropyltrimethylammonium bromide, and potassium iodide are mixed and added with acetonitrile, heated, and then cooled to room temperature, and the precipitated solid is collected and washed to obtain ND-SF-3; ND-SF-3 and potassium hexafluorophosphate are mixed and added with acetonitrile, heated, and then cooled to room temperature, and the solid is collected and washed to obtain the fluorescent probe. The fluorescent probe provided by this invention is simple to prepare, low in cost, highly sensitive, selective, and biocompatible; it can sensitively detect changes in mitochondrial viscosity in organisms.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis and detection technology, specifically relating to the preparation method and detection method of a fluorescent probe for detecting changes in mitochondrial viscosity in organisms. Background Technology

[0002] Mitochondrial viscosity is an important physical parameter reflecting the functional state of cells. When mitochondrial viscosity changes, it has a series of effects on cells. In terms of energy metabolism, it alters the rate of ATP synthesis, leading to insufficient cellular energy supply; in terms of substance transport, it affects the function of ADP and ATP transport enzymes, thus hindering metabolite exchange; and in terms of physiological diseases, it can lead to the development of neurodegenerative diseases, cardiovascular diseases, cancer, and other diseases.

[0003] Traditional viscosity detection methods generally suffer from limitations such as cumbersome sample pretreatment, insufficient sensitivity, and difficulty in real-time dynamic monitoring, failing to meet the needs of live cell and in vivo imaging. In contrast, fluorescent probe technology, due to its advantages of simple operation, high sensitivity, and real-time monitoring, has become an important tool for biological research and disease monitoring.

[0004] However, existing probes still face two major bottlenecks: first, the relatively short emission wavelength is easily affected by scattering and absorption from biological tissues, limiting signal penetration depth and detection accuracy; second, the small Stokes shift causes the emission spectrum to overlap with background fluorescence, significantly reducing the signal-to-noise ratio. This problem is particularly prominent in cell and tissue viscosity detection, severely restricting the practical application effectiveness of fluorescent probes. Therefore, developing novel fluorescent probes that combine long emission wavelengths and large Stokes shifts has significant theoretical guidance and application value for reducing background interference and improving the imaging quality of deep tissues. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method for preparing a fluorescent probe for detecting changes in mitochondrial viscosity in organisms and a method for detecting such changes.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention discloses a method for preparing a fluorescent probe for detecting changes in mitochondrial viscosity in organisms, comprising the following steps: S1. Triphenylbromoethylene and a five-membered heterocyclic compound containing aldehyde and boric acid groups were dissolved in an aqueous solution of isopropanol and potassium carbonate. The mixture was then stirred at room temperature for 60 min. A catalyst was then added, and the mixture was heated to 90 °C and reacted for 12 h. The mixture was then cooled to room temperature, and the isopropanol was removed by vacuum evaporation. Water was then added, and the mixture was finally extracted with dichloromethane. The organic phase was dried with anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by column chromatography to obtain a yellow powder, namely ND-SF-1, which is 5-(1,2,2-triphenylvinyl)thiophene-2-carboxaldehyde. S2. ND-SF-1 was mixed with 4-pyridine acetonitrile and then anhydrous ethanol was added. Piperidine was added dropwise and the temperature was raised to 78°C for reaction. The mixture was then cooled and refluxed for 12 h. After the reaction was completed, the mixture was cooled to room temperature, and the precipitated solid was collected by filtration. The solid was washed with ethyl acetate to obtain ND-SF-2. ND-SF-2 is (E)-2-(pyridin-4-yl)-4-(5-(1,2,2-triphenylvinyl)thiophen-2-yl)but-3-enonitrile. S3. ND-SF-2, 3-bromopropyltrimethylammonium bromide, and potassium iodide were mixed and then added to acetonitrile. The mixture was heated to 80°C and reacted for 8 h. After the reaction was completed, the mixture was cooled to room temperature, and the precipitated solid was collected. The solid was washed with ethyl acetate to obtain the red product ND-SF-3. ND-SF-3 is (E)-4-(1-cyano-3-(5-(1,2,2-triphenylvinyl)thiophen-2-yl)allyl)-1-(3-(trimethylamino)propyl)pyridine-1-bromoonium; S4. Mix ND-SF-3 and potassium hexafluorophosphate, add acetonitrile, heat to 80℃ and react for 8 h. After the reaction is complete, cool to room temperature, collect the precipitated solid, wash the solid with ethyl acetate to obtain a red product, which is the fluorescent probe for detecting changes in mitochondrial viscosity in organisms. The five-membered heteroaromatic compound containing an aldehyde group and a boric acid group is either 5-aldehyde-2-thiopheneboronic acid or 5-formyl-2-furanboronic acid; When the aldehyde-substituted heterocyclic boric acid is 5-aldehyde-2-thiopheneboronic acid, the probe is ND-SF, wherein the ND-SF is (E)-4-(1-cyano-3-(5-(1,2,2-triphenylvinyl)thiophene-2-yl)allyl)-1-(3-(trimethylamino)propyl)pyridine-1-hexafluorophosphate; When the aldehyde-substituted heterocyclic boric acid is 5-formyl-2-furanboronic acid, the probe is ND-FN, which is (E)-4-(1-cyano-3-(5-(1,2,2-triphenylvinyl)furan-2-yl)allyl)-1-(3-(trimethylamino)propyl)pyridine-1-hexafluorophosphate).

[0007] Further, in step S1, the molar ratio of the triphenylbromoethylene, the five-membered heteroaromatic compound containing aldehyde and boric acid groups is 1:1; the volume ratio of the isopropanol and potassium carbonate is 40:40; and the column chromatography uses ethyl acetate and petroleum ether in a volume ratio of 1:20 for purification.

[0008] Furthermore, during the reaction of triphenylbromoethylene, a five-membered heterocyclic compound containing aldehyde and boric acid groups, isopropanol, and potassium carbonate, N2 gas is continuously introduced for protection; the catalyst is 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride.

[0009] Further, in step S2, the molar ratio of ND-SF-1 to 4-pyridineacetonitrile is 1:1; and the volume of anhydrous ethanol is 40 mL.

[0010] Further, in step S3, the molar ratio of ND-SF-2, 3-bromopropyltrimethylammonium bromide, potassium iodide, and acetonitrile is 1:2:2, and the amount of acetonitrile is 40 mL.

[0011] Further, in step S4, the molar ratio of ND-SF-3 and potassium hexafluorophosphate is 1:1; the acetonitrile is 40 mL. Further, when the aldehyde-substituted heterocyclic boronic acid is 5-aldehyde-2-thiopheneboronic acid, the probe's structural formula is shown in Formula 1: Formula 1 When the aldehyde-substituted heterocyclic boric acid is 5-formyl-2-furanboronic acid, the structural formula of the probe is shown in Formula 2: Formula 2.

[0012] Further, probe ND-SF or probe ND-FN was dissolved in DMSO to prepare a 1×10⁻⁶ solution. -3 A stock solution of mol / L was prepared; then, PBS solution and glycerol were mixed at different volume ratios to prepare test systems of different viscosities for fluorescence testing, wherein the volume ratio of glycerol was 0-99%.

[0013] The beneficial effects of this invention are as follows: 1. The fluorescent probe provided by this invention is simple to prepare, low in cost, highly sensitive, selective, and biocompatible; it can sensitively detect changes in mitochondrial viscosity in organisms.

[0014] 2. The detection method provided by this invention is simple to operate, uses inexpensive solvents, and has convenient post-processing.

[0015] 3. This invention uses styrene as the fluorescent parent material and introduces thiophene or furan π-bridges and aldehyde groups through a Suzuki coupling reaction to construct a D-π-A structure to extend the emission wavelength. Subsequently, pyridine acetonitrile is linked through a Knoevenagel condensation reaction to further extend the conjugated system and introduce a molecular rotor structure. Finally, mitochondrial targeting groups and hexafluorophosphate ions are introduced through quaternization and ion exchange reactions, ultimately obtaining target probes ND-SF and ND-FN with large emission wavelengths, large Stokes shifts, and excellent water solubility.

[0016] Other advantages, objectives, and features of the invention will be set forth in the following description and will be apparent to those skilled in the art in some respects, or may be learned by practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0017] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 The synthetic routes for the fluorescent probes ND-SF and ND-FN of this invention are shown below; Figure 2 (a) is the proton NMR spectrum of the probe ND-SF; Figure 2 (b) is the carbon NMR spectrum of the probe ND-SF; Figure 2 (c) shows the mass spectrum of the probe ND-SF; Figure 3 The proton NMR spectrum of the probe ND-FN; Figure 4 The fluorescence intensity of probes ND-SF and ND-FN in solvents of different polarities; Figure 5 The fluorescence intensity of the probe ND-SF in systems with different viscosity; Figure 6 This is a graph showing the cytotoxicity of the probe ND-SF. Figure 7 Colocalization map of probe ND-SF; Figure 8 (a) Changes in mitochondrial viscosity in inflammatory cells were detected using the ND-SF probe; Figure 8 (b) is a comparison of the fluorescence intensity of the ND-SF probe; Figure 9 (a) Changes in mitochondrial viscosity in acutely inflamed mouse cells were detected using the ND-SF probe; Figure 9 (b) is a comparison of the fluorescence intensity of the probe ND-SF. Detailed Implementation

[0018] like Figures 1-9 As shown, this invention discloses a method for preparing a fluorescent probe for detecting changes in mitochondrial viscosity in organisms and a method for detecting such changes.

[0019] Example 1 S1. Triphenylbromoethylene (1 mmol), 5-aldehyde-2-thiopheneboronic acid (1 mmol), isopropanol (40 mL), and potassium carbonate aqueous solution (40 mL, 2 M) were placed in a three-necked flask equipped with a magnetic stirrer. The mixture was stirred at room temperature for 60 min under continuous N2 gas protection. After the reaction, a small amount of 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride (0.05 mmol) was added as a catalyst. The mixture was heated to 90 °C and reacted for 12 h. Then, the mixture was cooled to room temperature, and the isopropanol was removed by reduced pressure and rotary evaporation. Water was then added, and the mixture was extracted with dichloromethane (30 mL × 3). The organic phase was dried with Na2SO4, the solvent was evaporated, and the crude product was purified by column chromatography (ethyl acetate: petroleum ether = 1:20) to obtain a yellow powder, which is ND-SF-1. ND-SF-1 (1 mmol) and 4-pyridineacetonitrile (1 mmol) were added to a single-necked flask, 40 mL of anhydrous ethanol and two drops of piperidine were added, and the temperature was raised to 78 °C and then refluxed for 12 h. After the reaction was completed, the mixture was cooled to room temperature, the precipitated solid was collected by filtration, and the solid was washed with ethyl acetate to obtain ND-SF-2. ND-SF-2 (1 mmol), 3-bromopropyltrimethylammonium bromide (2 mmol), and potassium iodide (2 mmol) were added to a single-necked flask, 40 mL of acetonitrile was added, and the mixture was heated to 80 °C and reacted for 8 h. After the reaction was completed, the mixture was cooled to room temperature, the precipitated solid was collected, and the solid was washed with ethyl acetate to obtain the red product ND-SF-3. ND-SF-3 (1 mmol) and potassium hexafluorophosphate (1 mmol) were added to a single-necked flask, followed by 40 mL of acetonitrile. The mixture was heated to 80 °C and reacted for 8 h. After the reaction was complete, the mixture was cooled to room temperature, and the precipitated solid was collected. The solid was washed with ethyl acetate to obtain the red product ND-SF, as shown in the figure. Figure 2 As shown.

[0020] Example 2 Example 2: 5-Aldehyde-2-thiopheneboronic acid was replaced with 5-formyl-2-furanboronic acid, ultimately yielding the red product ND-FN, as shown below. Figure 3 As shown.

[0021] Testing and verification were conducted. The probes ND-SF and ND-FN were dissolved in DMSO to prepare a stock solution of 1×10⁻³ mol / L. PBS solution was then mixed with glycerol at different volume ratios (the glycerol volume ratio gradually increased from 0 to 99%) to prepare test systems of varying viscosities for fluorescence testing. Figure 4 As shown, the results indicate that the probe ND-SF exhibits good solvent selectivity. Figure 5 As shown, in the viscosity titration experiment, the fluorescence intensity of the ND-SF probe gradually increased with increasing viscosity, indicating that it has a highly sensitive viscosity response. Fluorescence spectra were measured at excitation wavelengths of 496 nm and 509 nm, with both Ex site and Emsite at 5 nm to ensure experimental repeatability.

[0022] Cytotoxicity assay: HepG2 cells were co-incubated with ND-SF probe at concentrations of 5 μM, 10 μM, 15 μM, and 20 μM for 24 h; a blank control group without probe was also incubated in parallel. Figure 6 As shown, even when HepG2 cells were incubated with the probe at a concentration of 20 μM for 24 h, the cell survival rate was still as high as 89%, indicating that it has low biotoxicity.

[0023] Mitochondrial colocalization experiments were performed using the probe ND-SF (10 μM) and the mitochondrial-targeting probe (200 μM). HepG2 cells were seeded on culture dishes and cultured at 37°C and 5% CO2 for 24 h. Then, 200 μM of MitoTracker Green dye (excitation wavelength 490 nm; emission wavelength 516 nm) and 10 μM of ND-SF probe (excitation wavelength 496 nm; emission wavelength 708 nm) were added, and the cells were incubated for 0.5 h. After washing with PBS, imaging was performed in different channels. Figure 7 As shown, PCC=0.9214 indicates that it has good mitochondrial targeting ability.

[0024] Detection of mitochondrial viscosity changes in inflammatory cells: HepG2 cells were pretreated with different concentrations of lipopolysaccharide (0 μM, 10 μM, 20 μM), and then co-incubated with a probe (10 μM) for 30 minutes to detect changes in mitochondrial viscosity during inflammation. Subsequently, HepG2 cells were pretreated with different concentrations of lipopolysaccharide 20 μM + apocynin (10 μM, 20 μM), and then co-incubated with a probe (10 μM) for 30 minutes to detect changes in mitochondrial viscosity during inflammation. The control group was only co-incubated with ND-SF (10 μM) for 30 minutes, and then mitochondrial viscosity changes were detected. Figure 8As shown, the probe can sensitively detect changes in intracellular mitochondrial viscosity.

[0025] Detection of mitochondrial viscosity changes in mice with acute inflammation: Group 1 mice received no treatment and underwent fluorescence imaging; Group 2 mice underwent fluorescence imaging after intraperitoneal injection of ND-SF (10 μM); Group 3 mice were induced with LPS (20 μM) intraperitoneally for 4 h to obtain an acute inflammation mouse model, and then injected with ND-SF (10 μM) for fluorescence imaging; Group 4 mice were induced with LPS (20 μM) intraperitoneally for 4 h to obtain an acute inflammation mouse model, and were first injected with apocynin (20 μM), then with ND-SF (10 μM) for fluorescence imaging. Figure 9 As shown, the probe can sensitively detect changes in mitochondrial viscosity in mice with acute inflammation.

[0026] The probe of this invention achieves a specific response to mitochondrial viscosity based on an intramolecular rotational restriction mechanism. The probe molecule contains multiple freely rotating benzene rings and carbon-carbon single bonds. In low-viscosity environments, these rotors can rotate rapidly, and excited-state energy is mainly dissipated through non-radiative pathways, resulting in weak fluorescence intensity. As the environmental viscosity increases, the solvent friction effect significantly enhances the resistance to rotor rotation, introducing an additional activation energy barrier for the dissipation of excited-state energy. When this activation energy exceeds the molecule's own rotational energy barrier, non-radiative transition pathways are effectively suppressed, and excited-state energy is released through radiative transition pathways, resulting in significant fluorescence enhancement. Therefore, fluorescence intensity is positively correlated with microenvironment viscosity.

[0027] Furthermore, the quaternary ammonium cations introduced into the probe molecules enable them to selectively accumulate at the negatively charged mitochondrial membrane potential, thereby achieving precise mitochondrial targeting. Through this design, probes ND-SF and ND-FN exhibit Stokes shifts of 212 nm and 196 nm, respectively, effectively avoiding background fluorescence interference and significantly improving the accuracy and signal-to-noise ratio of detection in complex biological systems.

[0028] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing a fluorescent probe for detecting changes in mitochondrial viscosity in organisms, characterized in that: Includes the following steps, S1. Triphenylbromoethylene and a five-membered heterocyclic compound containing aldehyde and boric acid groups were dissolved in an aqueous solution of isopropanol and potassium carbonate. The mixture was then stirred at room temperature for 60 min. A catalyst was then added, and the mixture was heated to 90 °C and reacted for 12 h. The mixture was then cooled to room temperature, and the isopropanol was removed by vacuum evaporation. Water was then added, and the mixture was finally extracted with dichloromethane. The organic phase was dried with anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by column chromatography to obtain a yellow powder, namely ND-SF-1, which is 5-(1,2,2-triphenylvinyl)thiophene-2-carboxaldehyde. S2. ND-SF-1 was mixed with 4-pyridine acetonitrile and then anhydrous ethanol was added. Piperidine was added dropwise and the temperature was raised to 78°C for reaction. The mixture was then cooled and refluxed for 12 h. After the reaction was completed, the mixture was cooled to room temperature, and the precipitated solid was collected by filtration. The solid was washed with ethyl acetate to obtain ND-SF-2. ND-SF-2 is (E)-2-(pyridin-4-yl)-4-(5-(1,2,2-triphenylvinyl)thiophen-2-yl)but-3-enonitrile. S3. ND-SF-2, 3-bromopropyltrimethylammonium bromide, and potassium iodide were mixed and then added to acetonitrile. The mixture was heated to 80°C and reacted for 8 hours. After the reaction was completed, the mixture was cooled to room temperature, and the precipitated solid was collected. The solid was washed with ethyl acetate to obtain the red product ND-SF-3. The ND-SF-3 is (E)-4-(1-cyano-3-(5-(1,2,2-triphenylvinyl)thiophen-2-yl)allyl)-1-(3-(trimethylamino)propyl)pyridine-1-bromoonium; S4. Mix ND-SF-3 and potassium hexafluorophosphate, add acetonitrile, heat to 80℃ and react for 8 h. After the reaction is complete, cool to room temperature, collect the precipitated solid, wash the solid with ethyl acetate to obtain a red product, which is the fluorescent probe for detecting changes in mitochondrial viscosity in organisms. The five-membered heteroaromatic compound containing an aldehyde group and a boric acid group is either 5-aldehyde-2-thiopheneboronic acid or 5-formyl-2-furanboronic acid; When the aldehyde-substituted heterocyclic boric acid is 5-aldehyde-2-thiopheneboronic acid, the probe is ND-SF, wherein the ND-SF is (E)-4-(1-cyano-3-(5-(1,2,2-triphenylvinyl)thiophene-2-yl)allyl)-1-(3-(trimethylamino)propyl)pyridine-1-hexafluorophosphate; When the aldehyde-substituted heterocyclic boric acid is 5-formyl-2-furanboronic acid, the probe is ND-FN, which is (E)-4-(1-cyano-3-(5-(1,2,2-triphenylvinyl)furan-2-yl)allyl)-1-(3-(trimethylamino)propyl)pyridine-1-hexafluorophosphate).

2. The method for preparing a fluorescent probe for detecting changes in mitochondrial viscosity in vivo according to claim 1, characterized in that: In step S1, the molar ratio of triphenylbromoethylene, the five-membered heteroaromatic compound containing aldehyde and boric acid groups is 1:1; the volume ratio of isopropanol and potassium carbonate is 40:40; and the column chromatography uses ethyl acetate and petroleum ether in a volume ratio of 1:20 for purification.

3. The method for preparing a fluorescent probe for detecting changes in mitochondrial viscosity in vivo according to claim 1, characterized in that: During the reaction of triphenylbromoethylene, a five-membered heterocyclic compound containing aldehyde and boric acid groups, isopropanol, and potassium carbonate, N2 gas is continuously introduced for protection; the catalyst is 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride.

4. The method for preparing a fluorescent probe for detecting changes in mitochondrial viscosity in vivo according to claim 3, characterized in that: In step S2, the molar ratio of ND-SF-1 to 4-pyridineacetonitrile is 1:1; the volume of anhydrous ethanol is 40 mL.

5. The method for preparing a fluorescent probe for detecting changes in mitochondrial viscosity in vivo according to claim 4, characterized in that: In step S3, the molar ratio of ND-SF-2, 3-bromopropyltrimethylammonium bromide, potassium iodide, and acetonitrile is 1:2:2, and the amount of acetonitrile is 40 mL.

6. The method for preparing a fluorescent probe for detecting changes in mitochondrial viscosity in vivo according to claim 5, characterized in that: In step S4, the molar ratio of ND-SF-3 to potassium hexafluorophosphate is 1:1; the amount of acetonitrile is 40 mL.

7. The method for preparing a fluorescent probe for detecting changes in mitochondrial viscosity in vivo according to claim 1, characterized in that: When the aldehyde-substituted heterocyclic boric acid is 5-aldehyde-2-thiopheneboronic acid, the structural formula of the probe is shown in Formula 1: Formula 1 When the aldehyde-substituted heterocyclic boric acid is 5-formyl-2-furanboronic acid, the structural formula of the probe is shown in Formula 2: Equation 2.

8. A method for detecting changes in mitochondrial viscosity in vivo, using the fluorescent probe as described in any one of claims 1-7, characterized in that: The probe ND-SF or probe ND-FN was dissolved in DMSO to prepare a 1×10⁻⁶ solution. -3 A stock solution of mol / L was prepared; then, PBS solution and glycerol were mixed at different volume ratios to prepare test systems of different viscosities for fluorescence testing, wherein the volume ratio of glycerol was 0-99%.

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