Phosphorescent probes for cell membrane imaging and methods of making and using the same

Abstract

The application belongs to the technical field of molecular probes, and particularly relates to a phosphorescent probe for cell membrane imaging and a preparation method and application thereof. The cell membrane imaging phosphorescent probe is M-Chol, M is Ru or Ir, a transition metal complex with a central ion of ruthenium or iridium is a luminophore, and cholesterol is a cell membrane targeting group. The phosphorescent probe has the advantages of long lifetime, two-photon excitation and good cell membrane targeting, can make up for the shortcomings of existing cell membrane targeting fluorescent probes, and becomes a powerful tool for studying the morphology and dynamic changes of cell membranes.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular probes, and relates to a phosphorescent probe for cell membrane imaging and a preparation method and application thereof. BACKGROUND

[0002] The cell membrane is an important barrier for protecting numerous organelles in cells from the surrounding environment, plays an important role in cell signal transduction and biomolecule transport, and is closely related to many physiological and pathological processes. Monitoring and analyzing the dynamic behavior of the cell membrane helps to reveal key processes such as cell activity and signal transduction pathways, and is crucial for application fields such as drug screening, early diagnosis and treatment of diseases, and control of signal transduction.

[0003] At present, the method for labeling the cell membrane based on a fluorescent probe is widely applied, the method can monitor the dynamic changes of the cell membrane in living cells in real time, is an important way for visualizing the cell membrane in situ, and provides key information for analyzing the function of the cell membrane.

[0004] Although the cell membrane probe with an organic group as a fluorescent emitter has advantages such as high fluorescence quantum yield, large molar extinction coefficient, and adjustable emission wavelength, the cell membrane probe has a small Stokes shift and is easily disturbed by excited light; the cell membrane probe has a short luminescence lifetime and is easily disturbed by short-lifetime strong background fluorescence in a living body, thereby affecting the accuracy of analysis, detection and imaging.

[0005] In addition to selecting a luminescent group, an effective targeting unit is also one of the key steps for developing a cell membrane fluorescent probe. The design of the membrane targeting unit mainly considers two key components of the cell membrane, namely, cell membrane proteins and phospholipid bilayers. The cell membrane protein labeling method is mainly based on fusion protein labeling, antibodies, small molecule ligands and aptamers. However, the probe design of the method is complex and the raw material cost is high. The cell membrane targeting probe with the phospholipid bilayer as the target unit takes an alkyl chain, cholesterol and tocopherol as the cell membrane targeting group. When the alkyl chain and tocopherol are selected as the cell membrane targeting group, the two are easily internalized into cells, and the residence time on the cell membrane is short, which is not conducive to long-term monitoring of the dynamic changes of the cell membrane.

[0006] The existing fluorescent probe for labeling the cell membrane has problems such as poor accuracy of analysis, detection and imaging, slow cell membrane labeling speed and poor anti-permeability, that is, easy internalization into cells to target other organelles. SUMMARY

[0007] In order to overcome the deficiencies of the prior art, the application provides a phosphorescent probe for cell membrane imaging and a preparation method and application thereof. The probe has a parent phosphor group of a Ru(II) or iridium(III) complex, has advantages of large Stokes shift, long luminescence lifetime, stable optical and thermodynamic properties and the like. Cholesterol is selected as a cell membrane targeting group, and the rigid structure of cholesterol is beneficial to long-term labeling on the cell membrane, and the probe can be kept on the cell membrane for 120 minutes without internalization. In addition, the probe has fast cell membrane targeting speed, and cell membrane imaging effect can be achieved only by incubation for 5 minutes, and has good penetration resistance, which is superior to existing commercial cell membrane dyes.

[0008] The above object of the application is achieved by the following technical solutions.

[0009] A first object of the application is to provide a phosphorescent probe for cell membrane imaging, which is a complex M-Chol with ruthenium or iridium as a central ion, M is Ru or Ir, and the molecular structure general formula is as follows:

[0010]

[0011] A second object of the application is to provide a synthesis method of the above-mentioned phosphorescent probe for cell membrane imaging, comprising the following steps:

[0012] S1. 4,4-dimethyl-2,2-bipyridine is used as a reactant, selenium dioxide is used as a catalyst, 1,4-dioxane is used as a solvent, and reflux stirring is carried out under argon protection conditions for 24 hours, then filtration and column chromatography purification are carried out to obtain a compound of formula 1.

[0013] S2. Formula 1 obtained in S1 and silver nitrate are used as reactants, sodium hydroxide is used as a base, ethanol and deionized water are used as solvents, and reaction is carried out at room temperature for 24 hours, then filtration is carried out, and the pH of the solution is adjusted to obtain a compound of formula 2.

[0014] S3. Formula 2 obtained in S2 and thionyl chloride are used as reactants, reflux stirring is carried out under argon protection conditions for 3 hours, then formula 3 is obtained after vacuum distillation, then formula 3 and cholesterol are used as reactants, anhydrous triethylamine is used as a base, anhydrous dichloromethane is used as a solvent, and reaction is carried out at room temperature for 24 hours, and then column chromatography purification is carried out to obtain a compound of formula 4.

[0015] S4. Ruthenium trichloride and bipyridine are used as reactants, N,N-dimethylformamide is used as a solvent, and reflux stirring is carried out under argon protection conditions for 8 hours, then the solution is cooled to room temperature, stirring is carried out for 30 minutes after adding acetone, and then overnight at 0°C, then filtration and washing are carried out to obtain a compound of formula 5;

[0016] S5. Iridium trichloride and phenylpyridine are used as reactants, ethylene glycol and water are used as solvents, and reflux stirring is carried out under argon protection conditions for 24 hours, and then formula 6 is obtained after filtration.

[0017] S6. Refluxing reaction with formula 4 and formula 5, formula 4 and formula 6 as reactants under the condition of argon protection, and purifying by column chromatography to obtain phosphorescent probe M-Chol, M is Ru or Ir;

[0018] The reaction formula is as follows:

[0019]

[0020] Further, in step S1, the molar ratio of the 4,4-dimethyl-2,2-bipyridine and selenium dioxide is 1:1.6.

[0021] Further, in step S1, the refluxing temperature is 106°C.

[0022] Further, in step S2, the molar ratio of formula 1, silver nitrate, and sodium hydroxide is 1:1:3.75.

[0023] Further, in step S2, the volume ratio of ethanol and deionized water is 1.6:1.

[0024] Further, in step S2, the pH is adjusted with hydrochloric acid; the concentration of the hydrochloric acid is 6 mol / L.

[0025] Further, in step S3, the molar ratio of formula 3, cholesterol, and triethylamine is 1:1:3.

[0026] Further, in step S3, the refluxing temperature is 80°C.

[0027] Further, in step S4, the molar ratio of ruthenium trichloride and bipyridine is 1:2.

[0028] Further, in step S5, the molar ratio of iridium trichloride and phenylpyridine is 1:2.

[0029] Further, in step S5, the volume ratio of ethylene glycol and deionized water is 3:1.

[0030] Further, in step S6, the molar ratio of formula 4 and formula 5 is 1:1; the molar ratio of formula 4 and formula 6 is 1:2.

[0031] The third object of the present application is to provide an application of the phosphorescent probe for cell membrane imaging prepared by the above-mentioned synthesis method in labeling cell membranes, which has high phosphorescent intensity in cell membrane imaging experiments, fast cell membrane targeting speed, and can achieve cell membrane imaging effect only by 5 minutes of incubation time. In addition, the probe also shows good penetration resistance and can remain on the cell membrane for 120 minutes without internalization, showing more excellent performance compared with the current commercial cell membrane dyes.

[0032] The present application uses a transition metal complex with ruthenium or iridium as a central ion as a luminophore, and uses cholesterol as a cell membrane targeting group, has the advantages of long lifetime, two-photon excitation and good cell membrane targeting, can make up for the shortcomings of existing cell membrane targeting fluorescent probes, and becomes a powerful tool for studying cell membrane morphology and dynamic changes.

[0033] Compared with the prior art, the present application has the following beneficial effects:

[0034] (1) Compared with traditional organic fluorescent probes, the Stokes shift of the transition metal complex of Ru(II) and iridium(III) is large, which can avoid the overlap of the emission spectrum and the excitation spectrum; the luminescence lifetime is long, which can eliminate the interference of background fluorescence through time-resolved fluorescence mode; and the optical and thermodynamic stability is strong, which is suitable for in-situ analysis.

[0035] (2) Compared with alkyl chains and tocopherols, cholesterol is selected as a cell membrane targeting group, and the special rigid structure of cholesterol is conducive to its long-term labeling on the cell membrane, and the probe can remain on the cell membrane for 120 minutes without internalization. Cell imaging results show that the probe can realize imaging of the cell membrane only by 5 minutes of incubation time, and has good penetration resistance, which is superior to existing commercial cell membrane dyes. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 The nuclear magnetic resonance hydrogen spectrum of the phosphorescent probe Ru-Chol prepared in Example 1;

[0037] Figure 2 The nuclear magnetic resonance carbon spectrum of the phosphorescent probe Ru-Chol prepared in Example 1;

[0038] Figure 3 The high-resolution mass spectrum of the phosphorescent probe Ru-Chol prepared in Example 1;

[0039] Figure 4 The nuclear magnetic resonance hydrogen spectrum of the phosphorescent probe Ir-Chol prepared in Example 1;

[0040] Figure 5 The nuclear magnetic resonance carbon spectrum of the phosphorescent probe Ir-Chol prepared in Example 1;

[0041] Figure 6 High resolution mass spectrum of phosphorescent probe Ir-Chol prepared in Example 1;

[0042] Figure 7 UV-Vis absorption spectrum and excitation-emission spectrum of phosphorescent probe Ru-Chol prepared in Example 1; wherein Figure a is the UV-Vis absorption spectrum of Ru-Chol, and Figure b is the phosphorescence excitation-emission spectrum of Ru-Chol;

[0043] Figure 8 UV absorption spectrum and excitation-emission spectrum of phosphorescent probe Ir-Chol prepared in Example 1; wherein Figure a is the UV-Vis absorption spectrum of Ir-Chol, and Figure b is the phosphorescence excitation-emission spectrum of Ir-Chol;

[0044] Figure 9 Fluorescence lifetime decay curve of phosphorescent probe M-Chol (M is Ru or Ir) prepared in Example 1; wherein Figure a is the fluorescence lifetime decay curve of Ru-Chol, and Figure b is the fluorescence lifetime decay curve of Ir-Chol;

[0045] Figure 10 Cell toxicity diagram of phosphorescent probe Ru-Chol for cell membrane imaging prepared in Example 1 on Hela cells;

[0046] Figure 11 Confocal imaging diagram of phosphorescent probe Ru-Chol for cell membrane imaging prepared in Example 1 on Hela cells. DETAILED DESCRIPTION

[0047] The present application will be described in detail below through specific examples, but does not limit the protection scope of the present application. Unless otherwise specified, the experimental methods used in the present application are conventional methods, and the experimental apparatus, materials, reagents, etc. used can be obtained from commercial channels.

[0048] Example 1

[0049] Example 1: Synthesis of phosphorescent probe M-Chol (M is Ru or Ir) for cell membrane imaging

[0050]

[0051] S1, 4,4-dimethyl-2,2-bipyridine (5.0 g, 27.5 mmol) and selenium dioxide (5.0 g, 45 mmol) were dissolved in 250 mL of 1,4-dioxane, stirred under argon protection conditions at reflux (106°C) for 24 h. After the reaction was completed, the black solid was removed by hot filtration, the solvent was removed by reduced pressure distillation, and the crude product was purified by silica gel column chromatography with pure ethyl acetate as the eluent to obtain white solid Formula 1 (3.16 g, 59%).

[0052]

[0053] S2, Formula 1 (3.16 g, 16 mmol) was dissolved in 160 mL of ethanol, 40 mL of silver nitrate (2.72 g, 16 mmol) aqueous solution was added, after stirring for 30 min, 60 mL of sodium hydroxide (2.4 g, 60 mmol) aqueous solution was added dropwise, and the reaction was carried out at room temperature for 24 h. After the reaction was completed, the solid was removed by filtration, ethanol was removed by distillation under reduced pressure, the solution was adjusted to pH 3.5 with hydrochloric acid, and white solid Formula 2 (2.0 g, 58%) was obtained by filtration.

[0054]

[0055] S3, Formula 2 (1.07 g, 5 mmol) was dissolved in 30 mL of thionyl chloride, and stirred at reflux (80°C) under argon protection for 3 h, and yellow solid Formula 3 was obtained by distillation under reduced pressure. Without any treatment, Formula 3 and cholesterol (1.93 g, 5 mmol) were dissolved in 30 mL of anhydrous dichloromethane, anhydrous triethylamine (2.08 mL, 15 mmol) was added, and stirred at room temperature for 24 h. The solvent was removed by distillation under reduced pressure, and the crude product was purified by silica gel column chromatography using dichloromethane / methanol (volume ratio 100 / 1) as the eluent to obtain yellow solid Formula 4 (0.58 g, 20%).

[0056]

[0057] S4, Ruthenium trichloride (1.58 g, 6 mmol), bipyridine (1.87 g, 12 mmol) were dissolved in 10 mL of N,N-dimethylformamide, and stirred at reflux (153°C) under argon protection for 8 h. After cooling to room temperature, 50 mL of acetone was added and stirred for 30 min, and then filtered at 0°C overnight. After washing with ether and water, black solid Formula 5 (2.5 g, 86%) was obtained.

[0058] S5, Iridium trichloride (352.6 mg, 1 mmol), bipyridine (310.4 mg, 2 mmol) were dissolved in ethylene glycol / water (volume ratio 3 / 1, 12 mL / 4 mL), and stirred at reflux (110°C) under argon protection for 24 h. After cooling to room temperature, yellow solid Formula 6 (482 mg, 90%) was obtained by filtration.

[0059] S6, formula 4 (116.6 mg, 0.2 mmol), and formula 5 (146 mg, 0.2 mmol) were dissolved in 40 mL of anhydrous ethanol and refluxed (80 °C) for 12 h under argon protection. The solvent was removed by vacuum distillation. The crude product was separated by silica gel column chromatography using acetonitrile / water (saturated potassium nitrate) (volume ratio 50 / 1). The eluent was collected, the solvent was evaporated to dryness, and 10 mL of acetonitrile was added. Excess potassium nitrate was then removed by filtration, and the solvent was evaporated to dryness. The resulting solid was dissolved in 2 mL of water, and 0.6 mL of saturated ammonium hexafluorophosphate aqueous solution was added. The mixture was filtered to obtain a brick-red solid phosphorescent probe Ru-Chol (220 mg, 86%). Formula 4 (116.6 mg, 0.2 mmol) and Formula 6 (107.2 mg, 0.1 mmol) were dissolved in a mixture of dichloromethane / methanol (volume ratio 2 / 1; 40 mL / 20 mL). The mixture was refluxed (65 °C) and stirred for 6 h under argon protection. After cooling to room temperature, ammonium hexafluorophosphate (0.32 g, 2 mmol) was added and stirred vigorously for 1 h. The mixture was filtered and the filtrate was evaporated by rotary evaporation. The crude product was purified by silica gel column chromatography using dichloromethane / methanol (volume ratio 100 / 1) as the eluent to obtain the orange phosphorescent probe Ir-Chol (108 mg, 44%).

[0060] The structure of the phosphorescent probe Ru-Chol synthesized in this embodiment was analyzed, and its proton NMR spectrum is shown in the figure. Figure 1 The carbon NMR spectrum is shown below. Figure 2 High-resolution mass spectrometry can be seen Figure 3 Furthermore, the structure of the phosphorescent probe Ir-Chol synthesized in this embodiment was analyzed, and its proton NMR spectrum is shown below. Figure 4 The carbon NMR spectrum is shown below. Figure 5 High-resolution mass spectrometry can be seen Figure 6 .

[0061] The phosphorescent probe M-Chol (M is Ru or Ir) synthesized in Example 1 was dissolved in 50 mM PBS buffer solution (pH = 7.4) to prepare a 30 μL test solution, and the UV-Vis absorption spectrum was measured. Figure 7 As shown in a, the maximum absorption peak of Ru-Chol is at 460 nm. The optimal absorption peak of Ir-Chol is at 360 nm. Figure 8 a).

[0062] The phosphorescent probe M-Chol (M is Ru or Ir) synthesized in Example 1 was dissolved in 50 mM PBS buffer solution (pH = 7.4) to prepare a 10 μL test solution, and the phosphorescence spectrum was measured. Figure 7 As shown in b, the maximum emission wavelength of Ru-Chol is 665 nm, and the Stokes shift is 205 nm; the maximum emission wavelength of Ir-Chol is 615 nm, and the Stokes shift is 255 nm.Figure 8 b) The phosphorescent probe has excellent luminescent performance, and the large Stokes shift avoids the overlap of the excitation spectrum and the emission spectrum, which is conducive to the collection of luminescent signals.

[0063] The phosphorescent probe M-Chol (M is Ru or Ir) synthesized in Example 1 was dissolved in a 50 mM PBS buffer solution (pH = 7.4) to prepare a 10 μL test solution for phosphorescent lifetime determination. By fitting, the phosphorescent lifetime of Ru-Chol was 257 ns ( Figure 9 a), and the phosphorescent lifetime of Ir-Chol was 131 ns ( Figure 9 b). It is shown that the phosphorescent probe has a long fluorescence lifetime, and the interference of background fluorescence can be eliminated by time-resolved fluorescence monitoring.

[0064] Cell toxicity was tested by MTT method. Hela cells were planted in a 96-well plate and placed in a 37°C incubator containing 5% CO2 / 95% air, and the cell density was 5 x 10 4 cells / mL. The culture medium was washed with PBS, and then PBS solutions containing different concentrations of Ru-Chol (0, 10, 20, 30, 50, 80, 100 μM) were added, respectively, and the cells were continued to be cultured in the 96-well plate at 37°C for 24 h. The culture solution was discarded, and the cells were washed with PBS for 3 times, then MTT (100 μL, 0.5 mg / mL) containing PBS solution was added, and the cells were continued to be cultured for 4 h. After removing the PBS solution containing MTT, 100 μL of DMSO was added to dissolve the cells in each well, and finally the absorbance of each well at 490 nm was determined by an enzyme marker, and the cell activity was calculated. As shown in Figure 10 even if the concentration of Ru-Chol reaches 100 μM, the survival rate of Hela cells is still above 85%, indicating that the probe has low cytotoxicity and can be used for imaging research in cells and living organisms.

[0065] Application Example

[0066] The phosphorescent probe Ru-Chol synthesized in Example 1 was subjected to cell imaging experiment, as shown in Figure 11 Ru-Chol at 20 μM successfully realized cell membrane imaging effect within 5 minutes of incubation time, and was labeled on the cell membrane for up to 120 minutes, indicating that the probe can quickly stain the cell membrane and has good anti-permeability ability, and can be kept on the cell membrane for 2 hours without internalization.

[0067] The above-described embodiments are merely preferred embodiments of the present application, but are not all the embodiments that can be implemented by the present application. Any obvious modifications made by those skilled in the art to the present application without departing from the principles and spirit of the present application should be considered to fall within the scope of protection of the claims of the present application.

Claims

1. A phosphorescent probe for cell membrane imaging, characterized by, The phosphorescent probe for cell membrane imaging is a complex M-Chol with ruthenium or iridium as central ion, M is Ru or Ir; the molecular structure general formula is as follows: 。 2. The method for synthesis of phosphorescent probe for cell membrane imaging according to claim 1, characterized in that, The method comprises the following steps: S1. 4,4-dimethyl-2,2-bipyridine as a reactant, selenium dioxide as a catalyst, 1,4-dioxane as a solvent, reflux stirring under argon protection for 24 h, filtration, column chromatography purification to obtain the compound of formula 1; S2. The compound of formula 1 obtained in S1 and silver nitrate as reactants, sodium hydroxide as a base, ethanol and deionized water as solvents, reaction at room temperature for 24 h, filtration, adjusting the pH of the solution to obtain the compound of formula 2; S3. The compound of formula 2 obtained in S2 and thionyl chloride as reactants, reflux stirring under argon protection for 3 h, vacuum distillation to obtain the compound of formula 3, then the compound of formula 3 and cholesterol as reactants, anhydrous triethylamine as a base, anhydrous dichloromethane as a solvent, reaction at room temperature for 24 h, column chromatography purification to obtain the compound of formula 4; S4. Ruthenium trichloride and bipyridine as reactants, N,N-dimethylformamide as a solvent, reflux stirring under argon protection for 8 h, cooling to room temperature, stirring with acetone for 30 min, then overnight at 0 ℃, filtration and washing to obtain the compound of formula 5; S5. Iridium trichloride and phenylpyridine as reactants, ethylene glycol and water as solvents, reflux stirring under argon protection for 24 h, filtration to obtain the compound of formula 6; S6. The compound of formula 4 and the compound of formula 5, the compound of formula 4 and the compound of formula 6 as reactants, reflux reaction under argon protection, column chromatography purification to obtain the phosphorescent probe M-Chol, M is Ru or Ir; The reaction formula is as follows: 。 3. The method for synthesizing the phosphorescent probe for cell membrane imaging as described in claim 2, characterized in that, In step S1, the molar ratio of 4,4-dimethyl-2,2-bipyridine to selenium dioxide is 1:1.

6.

4. The method for synthesizing the phosphorescent probe for cell membrane imaging as described in claim 2, characterized in that, In step S2, the molar ratio of the compound of formula 1, silver nitrate and sodium hydroxide is 1:1:3.

75.

5. The method for synthesizing the phosphorescent probe for cell membrane imaging as described in claim 2, characterized in that, In step S3, the molar ratio of the compound of formula 3, cholesterol and triethylamine is 1:1:

3.

6. The method for synthesizing the phosphorescent probe for cell membrane imaging as described in claim 2, characterized in that, In step S4, the molar ratio of ruthenium trichloride to bipyridine is 1:

2.

7. The method for synthesizing the phosphorescent probe for cell membrane imaging as described in claim 2, characterized in that, In step S5, the molar ratio of iridium trichloride to phenylpyridine is 1:

2.

8. The method of synthesis of phosphorescent probe for cell membrane imaging as claimed in claim 2, wherein the step of In S1, the reflux temperature is 106 ℃; in step S3, the reflux temperature is 80 ℃; in step S6, the molar ratio of the compound of formula 4 to the compound of formula 5 is 1:1; the molar ratio of the compound of formula 4 to the compound of formula 6 is 2:

1.

9. The method for synthesizing the phosphorescent probe for cell membrane imaging as described in claim 2, characterized in that, In step S2, the volume ratio of ethanol to deionized water is 1.6:1; in step S5, the volume ratio of ethylene glycol to deionized water is 3:

1.

10. The phosphorescent probe for cell membrane imaging prepared by the synthesis method of the phosphorescent probe for cell membrane imaging according to any one of claims 2-9 for use in the preparation of a cell membrane labeling reagent.

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