A fluorescent probe for detecting lysosomal viscosity and oxidative stress levels, its preparation method and application

Abstract

This invention discloses a fluorescent probe for detecting lysosomal viscosity and oxidative stress levels, its preparation method, and its application, relating to the field of fine chemical technology. The fluorescent probe has the structure shown in Formula I. By introducing pyrazolone groups into rhodamine-based fluorophores, this invention achieves targeted recognition of lysosomes, enabling simultaneous detection of lysosomal viscosity and oxidative stress levels. Tests show that the lysosomal functional imaging agent provided by this invention has many advantages such as near-infrared emission, wash-free operation, good biocompatibility, and high signal-to-noise ratio, enabling precise localization of lysosomes in cells, thereby obtaining high-fidelity information on lysosomal viscosity and oxidative stress levels.

Description

Technical Field

[0001] This invention relates to the field of fine chemical technology, specifically to a fluorescent probe for detecting lysosomal viscosity and oxidative stress levels, its preparation method, and its application. Background Technology

[0002] Lysosomes play a crucial role in multiple cellular processes, such as biomolecule degradation, apoptosis, cell signaling, cellular metabolic regulation, and plasma membrane repair. Lysosomal dysfunction is critical to the pathology of neurodegenerative diseases, aging, and cancer, making lysosomes a potential therapeutic target. Under physiological conditions, the pH of lysosomes is around 4.5. In cases of lysosomal dysfunction, macromolecules cannot be degraded and accumulate excessively within the lysosome, ultimately leading to significant changes in lysosomal viscosity. Lysosomes play a key role in the breakdown of iron-containing macromolecules; therefore, they have a high iron content. Ferrous iron can react with hydrogen peroxide via the Fenton reaction to form ferrous iron and harmful hydroxyl radicals (·OH). The acidic pH of lysosomes and the presence of reducing agents such as cysteine ​​provide a favorable environment for the Fenton reaction, and under oxidative stress, hydrogen peroxide readily diffuses from the cytoplasm into the lysosome. Furthermore, lysosomes typically lack reductases such as catalase or glutathione peroxidase, which exacerbates the potential for reactive iron-induced oxidative stress damage in lysosomes. Increasing evidence suggests that the degradation function of lysosomes is closely regulated by oxidative stress; therefore, monitoring the dynamic changes of reactive oxygen species (ROS) in lysosomes is crucial for determining their physiological functions and the pathogenesis of related diseases.

[0003] Fluorescence imaging offers advantages such as high spatiotemporal resolution, high sensitivity, and good selectivity, showing great potential for real-time monitoring of intracellular bioactive small molecules and key parameters related to the cellular microenvironment (pH, polarity, and viscosity). However, existing lysosomal probes can only detect single functions of lysosomes and cannot reflect the interactions and correlations between lysosomal functions, further limiting the study of lysosomal mechanisms.

[0004] Therefore, there is an urgent need to improve this shortcoming. This invention aims to improve the existing structure and its deficiencies, and provides a fluorescent probe for detecting lysosomal viscosity and oxidative stress levels, as well as its preparation method and application. Summary of the Invention

[0005] The purpose of this invention is to provide a fluorescent probe for detecting lysosomal viscosity and oxidative stress levels, as well as its preparation method and application, to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a fluorescent probe for detecting lysosomal viscosity and oxidative stress levels, its preparation method and application, the structure of which is shown in Formula I:

[0007]

[0008] In Formula I, R1 is independently selected from any one of the groups shown in Formulas II-VIII;

[0009]

[0010] The curve marks the substitution positions.

[0011] Furthermore, R2 is independently selected from any one of hydrogen (-H), methyl (-CH3), and phenyl (-C6H5).

[0012] Furthermore, R3 is independently selected from either methyl (-CH3) or ethyl (-CH2CH3).

[0013] Furthermore, R1 is the group shown in Formula IV, R2 is any one of hydrogen (-H) and phenyl (-C6H5), and R3 is methyl (-CH3).

[0014] Furthermore, a method for preparing a fluorescent probe for detecting lysosomal viscosity and oxidative stress levels, applied to the preparation of the aforementioned fluorescent probe for detecting lysosomal viscosity and oxidative stress levels, is characterized by the following preparation steps:

[0015] The steps include: preparing the target compound by a nucleophilic substitution reaction between the compound shown in Formula XIII and the compound shown in Formula XIV.

[0016]

[0017] Further, the target compound, namely the compound shown in Formula I, was prepared by reacting 1 equivalent of the compound shown in Formula IX with 1.56 equivalents of the corresponding pyrazolone X in an organic solvent such as acetonitrile for 20 minutes to 2 hours; after the reaction was completed, the fluorescent dye with the structure shown in Formula I was obtained by column chromatography.

[0018] Furthermore, the fluorescent probes described for detecting lysosomal viscosity and oxidative stress levels are used as fluorescent markers on cells.

[0019] Furthermore, the fluorescent probes used to detect lysosomal viscosity and oxidative stress levels are applied in in vivo imaging.

[0020] This invention provides a fluorescent probe for detecting lysosomal viscosity and oxidative stress levels, its preparation method, and its application, offering the following advantages: By introducing pyrazolone groups into rhodamine-based fluorophores, targeted recognition of lysosomes is achieved, enabling simultaneous detection of lysosomal viscosity and oxidative stress levels. Tests show that the lysosomal functional imaging agent provided by this invention possesses numerous advantages, including near-infrared emission, wash-free operation, good biocompatibility, and a high signal-to-noise ratio. It can achieve precise localization of lysosomes within cells, thereby obtaining high-fidelity information regarding lysosomal viscosity and oxidative stress levels. Attached Figure Description

[0021] Figure 1 This is a single-crystal configuration diagram of dye I-1 of the present invention;

[0022] Figure 2 The fluorescence spectra of dye I-2 of the present invention in phosphate buffer solutions at different pH values ​​are shown.

[0023] Figure 3 The fluorescence lifetime spectra of dye I-2 of the present invention in ethanol / glycerol mixed solution systems with different ratios are shown.

[0024] Figure 4 The fluorescence spectra of dye I-2 of the present invention after incubation with different concentrations of ·OH in a phosphate buffer / methanol mixed solution system;

[0025] Figure 5 This is a biocompatibility characterization diagram of dye I-2 of the present invention;

[0026] Figure 6 This is a colocalization map of dye I-2 of the present invention on lysosomes in cells. Detailed Implementation

[0027] like Figures 1-6 As shown, a fluorescent probe for detecting lysosomal viscosity and oxidative stress levels, wherein R1 is independently selected from any one of the groups shown in Formulas II-VIII;

[0028] R2 is independently selected from any one of hydrogen (-H), methyl (-CH3), and phenyl (-C6H5);

[0029] R3 is independently selected from either methyl (-CH3) or ethyl (-CH2CH3);

[0030] In a further preferred embodiment, R1 is the group shown in Formula IV, R2 is one of hydrogen (-H) and phenyl (-C6H5), and R3 is methyl (-CH3);

[0031] A further preferred form of R2 is phenyl;

[0032] The present invention provides a method for preparing the compound shown in Formula I. The main specific steps of the method are: reacting 1 equivalent of the compound shown in Formula IX with 1.56 equivalents of the corresponding pyrazolone X in an organic solvent such as acetonitrile (reaction medium) for 20 minutes to 2 hours to obtain the target product (the compound shown in Formula I).

[0033] The present invention will be further illustrated below through examples, the purpose of which is solely to provide a better understanding of the invention. Therefore, the examples given do not limit the scope of protection of the present invention.

[0034] Example 1

[0035] 1. Synthesis of Dye I-1

[0036]

[0037]

[0038] In a 50 mL single-necked flask, 100 mg (0.25 mmol) of IX-1, 3-methyl-2-pyrazolin-5-one (39 mg, 0.39 mmol), and 10 mL of acetonitrile were added. The reaction was carried out at room temperature for 0.5 h. The reaction solvent was removed by rotary evaporation, and the mixture was separated by column chromatography with a mobile phase ratio of DCM:MeOH = 30:1. 69 mg of a dark green solid of I-1 was obtained, with a yield of 57%.

[0039] 1 H NMR (400Hz, DMSO-d6, ppm): δ0.55(s,6H,-SiCH3),1.21(t,12H,-CH2CH3),1.91(s,3H,-CH3),3.69(d,8H,-CH2 CH3),6.90-6.93(dd,2H,J1=9.6Hz,J2=2.8Hz,Ph-H),7.34(d,J=2.8Hz,2H,Ph-H),7.46(d,J=9.6Hz,2H,Ph-H).

[0040] Mass spectrometry(ESI positive ion mode,m / z):[M] + calcd for C 27 H 37 N4OSi + ,461.2737,found,461.2733.

[0041] 2. Synthesis of Dye I-2

[0042]

[0043] In a 50 mL single-necked flask, 100 mg (0.25 mmol) of IX-1, 69 mg (0.39 mmol) of X-2, and 10 mL of acetonitrile were added. The reaction was allowed to proceed for 0.5 h. The reaction solvent was removed by rotary evaporation, and the mixture was separated by column chromatography with a mobile phase ratio of DCM:MeOH = 30:1. 106 mg of I-2 as a reddish-brown solid was obtained, with a yield of 76%.

[0044] 1 H NMR (400Hz, CDCl3, ppm): δ0.46 (s, 6H, -SiCH3), 1.18 (t, 12H, J = 7.2Hz, -CH2CH3), 1. 85(s,3H,-CH3),3.41(t,8H,J=7.2Hz,-CH2CH3),6.62-6.64(dd,2H,J1=8.8Hz,J2=2 .4Hz,Ph-H),6.86(d,2H,J=2.4Hz,Ph-H),7.11(t,1H,J=7.2Hz,Ph-H),7.35(t,2H,J =8.4Hz, Ph-H), 7.72 (d, 2H, J = 8.4Hz, Ph-H), 7.97 (d, 2H, J1 = 8.8Hz, J2 = 1.2Hz, Ph-H).

[0045] Mass spectrometry(ESI positive ion mode,m / z):[M+H] + calcd forC 33 H 41 N4OSi,537.3050,found,537.3049.

[0046] Example 2

[0047] Single crystal configuration of dye I-1

[0048] In a clean glass bottle, 4 mg of dye I-1 prepared in Example 1 was dissolved in 1 mL of dichloromethane (analytical grade), a good solvent. Hexane (analytical grade), a poor solvent, was slowly added along the bottle wall, layering on top of the good solvent. The upper layer was observed to be colorless and transparent, while the lower layer showed the color of the dissolved sample. The bottle was sealed with plastic wrap and a rubber band and placed in a refrigerator or quiet place to allow the solvent to evaporate slowly. The single-crystal configuration of dye I-1 was obtained as follows. Figure 1 As shown.

[0049] Example 3

[0050] Absorption and fluorescence spectra of dye I-2 at different pH values

[0051] The dye I-2 prepared in Example 1 was dissolved in analytical grade dimethyl sulfoxide to prepare a 1 mM test stock solution. Then, phosphate buffer solutions with different pH values ​​were prepared as test system solutions. 20 μL of the above stock solution was diluted to 2 mL of the test system solution, mixed thoroughly to obtain a final concentration of 10 μM test solution, and its fluorescence spectrum was measured in an optical quartz cuvette (10 × 10 mm). Figure 2 As shown, with 650 nm as the excitation wavelength, the maximum emission peak of dye I-2 is located at 680 nm in the near-infrared region; and the fluorescence of dye I-2 increases with decreasing pH value, exhibiting the characteristic of acidic fluorescence response, thus it can be localized to lysosomes.

[0052] Example 4

[0053] Dye I-2 is used for viscosity testing

[0054] The dye I-2 prepared in Example 1 was dissolved in analytical grade acetonitrile to prepare 1.0 × 10⁻⁶ ppm. -3 A stock solution of M was prepared. Then, 2 mL of ethanol / glycerol mixed solutions with different ratios were prepared. 20 μL of the above stock solution was added to the prepared ethanol / glycerol mixed solvent, mixed thoroughly, and then transferred to an optical quartz cuvette (10 × 10 mm) to test its fluorescence lifetime. Figure 3 As shown, with 650 nm as the excitation wavelength, the fluorescence lifetime of dye I-2 at 680 nm gradually increases with the increase of the viscosity of the test system, exhibiting viscosity-responsive characteristics. Therefore, it can be used to detect the viscosity in lysosomes.

[0055] Example 5

[0056] Dye I-2 is used for reactive oxygen species detection.

[0057] The dye I-2 prepared in Example 1 was dissolved in analytical grade acetonitrile to prepare 1.0 × 10⁻⁶ ppm. -3 A stock solution of M was prepared. Then, a 2 mL phosphate buffer / methanol (9:1, v / v, pH=5) mixed solution was prepared. 20 μL of the above stock solution was added to the prepared phosphate buffer / methanol mixed solvent, mixed thoroughly, and then transferred to an optical quartz cuvette (10×10 mm). The mixture was incubated with different concentrations of ·OH at 37℃ for 4 h, and then its fluorescence spectrum was measured. Figure 4 As shown, with 460 nm as the excitation wavelength, after gradually adding ·OH, the original emission at 680 nm of dye I-2 gradually decreased, and a new emission peak at 520 nm was generated. The two emission peaks had almost no spectral overlap, indicating that dye I-2 has the characteristic of responding to reactive oxygen species, and therefore can be used to detect the oxidation level in lysosomes.

[0058] Example 6

[0059] Biocompatibility test of dye I-2

[0060] The cytotoxicity of dye I-2 was tested using the standard MTT assay. The specific experimental steps are as follows: First, RAW 264.7 cells were seeded into 96-well plates at a cell density of 1 × 10⁻⁶ cells / well. 4 Cells were cultured overnight in a cell culture incubator. Different concentrations of dye I-2 (0-128 μM, 100 μL / well) were added to different wells, and DMSO diluted in culture medium (0.5%, 100 μL / well) served as a negative control. Cells were cultured in a cell culture incubator for 24 h, and then 10 μL of MTT solution (5 mg / mL) was added to each well. After 4 hours of incubation, the liquid in the 96-well plate was removed, and DMSO (100 μL / well) was added. Finally, the absorbance was measured using a microplate reader. Figure 5 The cytotoxicity data showed that when the concentration of dye I-2 was increased to 128 μM, the cell survival rate was approximately 88%, indicating that dye I-2 has very low cytotoxicity.

[0061] Example 7

[0062] Colocalization experiment of dye I-2 on lysosomes in cells

[0063] To investigate the intracellular distribution of dye I-2, a co-localization experiment was conducted on its subcellular organelles. The commercially available dye Lyso-Tracker Green was selected as the gold-labeled dye for co-localization in HeLa cells. The specific experimental steps are as follows: HeLa cells were incubated with the dye I-2 stock solution or the Lyso-Tracker Green stock solution from Example 2 at 37°C for 30 min (final concentration of dye I-2 was 10 μM, and final concentration of dye Lyso-Tracker Green was 1 μM). Cell imaging was then performed using a confocal microscope. The imaging results are shown below. Figure 6 As shown, dye I-2 clearly fluorescently labeled lysosomes, and the localization results were essentially consistent with those of organelles labeled by commercially available lysosomal green fluorescent probes (Lyso-Tracker Green). This demonstrates that dye I-2 can accurately localize lysosomes, with a colocalization Pearson coefficient of up to 0.90 with the lysosomal green fluorescent probe.

[0064] The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A fluorescent probe for detecting lysosomal viscosity and oxidative stress levels, characterized in that, Its structure is shown in Equation I: In Formula I, R1 is independently selected from any one of the groups shown in Formulas II-VIII; The curve marks the substitution positions; R2 is independently selected from any one of hydrogen, methyl, and phenyl; R3 is independently selected from either methyl or ethyl.

2. The fluorescent probe for detecting lysosomal viscosity and oxidative stress levels according to claim 1, characterized in that, R1 is the group shown in Formula IV, R2 is either hydrogen or phenyl, and R3 is methyl.

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