CN117088881A - Azabicyclo-naphthalene fluorescent molecular probe and preparation and application thereof - Google Patents

Azabicyclo-naphthalene fluorescent molecular probe and preparation and application thereof Download PDF

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CN117088881A
CN117088881A CN202311025550.4A CN202311025550A CN117088881A CN 117088881 A CN117088881 A CN 117088881A CN 202311025550 A CN202311025550 A CN 202311025550A CN 117088881 A CN117088881 A CN 117088881A
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邹宏斌
李进彪
劳嘉欣
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Zhejiang University ZJU
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Abstract

The invention provides an azabicyclo-naphthalene fluorescent molecular probe, and preparation and application thereof. The synthesis method of the azadicyclopentanaphthalene derivative is simple and convenient to operate. The synthesized fluorescent probe has large Stokes shift, narrow half-peak width, dual-state emission and aggregation-induced fluorescence properties. The fluorescent probe molecules can be specifically combined with different organelles in cells, are taken up by the cells and are gathered in different organelles such as cell membranes, lipid drops, mitochondria or lysosomes, and emit fluorescent signals under the excitation of ultraviolet light, so that the marking of different organelles in the cells is completed, and the fluorescent imaging of the organelles is realized.

Description

Azabicyclo-naphthalene fluorescent molecular probe and preparation and application thereof
Technical Field
The invention belongs to the field of new material fluorescent probes, and in particular relates to an azabicyclo-naphthalene fluorescent molecular probe and preparation and application thereof, which are suitable for imaging different organelle markers in cells.
Background
Organelles are the fundamental structural basis for the normal operation and functioning of cells, whose activities are closely related to life processes. For example, lipid droplets are organelles of lipid storage and metabolism in cells, whose activities are closely related to lipid storage metabolism, signal transduction, and apoptosis, and dyskinesias of lipid droplets have been shown to be closely related to various diseases such as viral infection, inflammation, obesity, and cancer. Cell membrane is a barrier for the exchange of substances inside and outside cells, the main components of which are proteins and lipids, and is a structural basis for maintaining the stability of intracellular environment and the signal transmission between cells, and at the same time, cell membrane fluorescence imaging is the most powerful means for dividing cell boundaries. Mitochondria are the primary sites of peroxidation phosphorylation and lipid oxidation reactions in eukaryotic cells, providing energy for cellular activities, and mitochondrial probes can be used to study mitochondrial function or its effects on cellular metabolism. Lysosomes can be combined with cell membranes or organelle membranes to participate in the processes of biological molecule degradation, apoptosis, signal transduction and the like in cells. Therefore, the development of specific organelle probes and their use for localization and tracking of intracellular organelles is of great interest.
Conventional fluorescent probes typically have a large conjugated plane and exhibit Aggregation-induced Quenching (ACQ) properties. These probes suffer from drawbacks such as small stokes shift, large emission half-width, background interference, and the like, which when used as organelle probes, tend to result in incomplete imaging of the organelle and loss of information. 2001, a class of molecules with Aggregation-Induced Emission (AIE) properties is reported, which has a completely opposite luminescence characteristic to ACQ-based fluorescent materials, and is characterized by not emitting fluorescence in a dissolved state, and exhibiting strong fluorescence after Aggregation. The AIE molecule solves the difficult problem of aggregation-induced quenching of the traditional fluorescent molecules during use, thereby widening the application field of fluorescent materials. In recent years, new fluorescent materials with solution-solid two-state Emission (DSE) have attracted attention from many researchers, and DSE-like fluorescent molecules have a wider range of applications. Compared with the traditional ACQ fluorescent molecules, the AIE fluorescent molecules and DSE fluorescent molecules have greater advantages in organelle imaging, such as good biocompatibility, high brightness, strong specificity and good light stability. In addition, DSE molecules can achieve fluorescence imaging of organelles over a wide range of low to high concentrations. Thus, an increasing number of organic AIE or DSE small molecules have been developed as specific organelle fluorescent probes.
Currently, there are challenges still faced in developing specific organelle fluorescent probes with AIE or DSE properties, such as: 1. the synthesis steps are complex, and most organelle probes need different positioning groups and are synthesized from scratch; 2. the half-width of fluorescence emission is large, signals of different probes are mutually interfered, and the method is not suitable for multiple labeling in complex cell environments; 3. the stokes shift is small and the excitation wavelength interferes with the signal at the emission wavelength.
Disclosure of Invention
Aiming at the defects or improvement demands of the prior art, the invention provides an azadicyclopentanaphthalene fluorescent molecular probe, which is a novel azadicyclopentanaphthalene fluorescent molecular probe, and the structure of the azadicyclopentanaphthalene derivative is as follows:
the fluorescent molecular probe I-1 provided by the invention has DSE property, the probes I-2, I-3 and I-4 have AIE property, different probes have specific targeting effect on different organelles, and corresponding fluorescent signals are generated in different organelles at different emission wavelengths under the action of excitation light, so that organelle imaging is realized. Wherein, I-1 can target lipid drops in cells, the excitation wavelength is 395nm, the emission wavelength is 520-580nm, and the lipid drops show orange fluorescence; i-2 can target cell membranes in cells, has an excitation wavelength of 374nm and an emission wavelength of 480-540nm, and shows green fluorescence; i-3 can target mitochondria in cells, has an excitation wavelength of 372nm and an emission wavelength of 480-540nm, and shows green fluorescence; i-4 can target lysosomes in cells, excitation wavelength is 369nm, emission wavelength is 480-540nm, and the lysosomes are green fluorescence.
The fluorescent molecular probe is DSE or AIE molecule, and has the advantages of large Stokes shift, strong organelle specificity, high fluorescence intensity and the like.
The invention also aims to provide a preparation method of the azabicyclo-naphthalene fluorescent molecular probe, which is realized by the following steps:
a) In pyridine, 4-tertiary butyl benzoyl chloride and imidazole generate M1 under the catalysis of triethylamine;
b) In a mixed solvent of acetonitrile and dichloromethane, the intermediate M1 reacts with bromoacetonitrile under the catalysis of cesium carbonate to generate an intermediate M2;
c) In tetrahydrofuran, the intermediate M2 generates a compound I-1 under the catalysis of potassium tert-butoxide;
d) In acetonitrile, the compound I-1 reacts with different iodides to respectively obtain the compounds I-2, I-3 and I-4.
The synthetic route is as follows:
in step a), the preferred conditions are: imidazole (0.68 g,0.01 mol) was dissolved in 4 mL of anhydrous pyridine under nitrogen protection, and triethylamine (4.2 mL,0.03 mol) was added, the reaction mixture was cooled with an ice bath, and 4-tert-butylbenzoyl chloride (5.88 g,0.03 mol) was added dropwise under the ice bath, then the mixture was stirred at room temperature for 2 hours, aqueous NaOH (40%, w/w,2 mL) was carefully added, and the mixture was refluxed for 1 hour, the reaction mixture was poured into cold water (20 mL) and stirred for 1 hour, a solid was filtered, and the solid was washed three times with 20mL of water and dried at 70 ℃ to obtain an off-white intermediate M1.
In step b), the preferred conditions are: intermediate M1 (0.912 g,4.0 mmol) was dissolved in a mixed solvent of 10 ml acetonitrile and 10 ml dichloromethane, cesium carbonate (1.564 g,4.8 mmol) was added at room temperature and reacted with stirring for 15 minutes, then bromoacetonitrile (0.571 g,4.8 mmol) was added dropwise, the mixture was reacted at room temperature with stirring for 2 hours again, the reaction solution was filtered and the obtained liquid was concentrated under reduced pressure, and the obtained residue was recrystallized in ethanol to obtain intermediate M2.
In step c), the preferred conditions are: intermediate M2 (0.80 g,3 mmol) was dissolved in 10 ml of anhydrous tetrahydrofuran under nitrogen protection, potassium tert-butoxide (0.67 g,6 mmol) was added at room temperature, the mixture was stirred at room temperature for 30 minutes, the reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give yellow solid I-1.
In step d), the preferred conditions are: compound I-1 (50 mg,0.1 mmol) and 1-iodooctadecane (77 mg,0.2 mmol) or (4-iodobutyl) triphenylphosphonium iodide (114 mg,0.2 mmol) or 4- (2-iodoethyl) morpholine (48 mg,0.2 mmol) were dissolved in 5 ml acetonitrile, the mixture was placed in a pressure-resistant tube and stirred at 120℃for 12 hours, the reaction mixture was then concentrated under reduced pressure, 5 ml ethyl acetate was added to the residue and sonicated for 30 minutes, and the resulting solid was filtered and rinsed twice with 5 ml ethyl acetate to give yellow solids I-2, I-3 and I-4, respectively.
It is a further object of the present invention to provide the use of said azabicyclo-naphthalene-based fluorescent molecular probes for fluorescent labeling and imaging of organelles, wherein said organelles are cell membranes, lipid droplets, mitochondria and lysosomes.
In a medium containing living cells (Dulbecco modified medium, DMEM), the fluorescent molecular probe of the present invention is directly added or a solution of the fluorescent molecular probe of the present invention dissolved in a solvent such as dimethyl sulfoxide, 1, 3-propanediol, physiological saline, etc., is taken up by the cells and aggregated in different organelles such as cell membranes, lipid droplets, mitochondria or lysosomes, and fluorescent signals are emitted under ultraviolet light excitation, thereby completing labeling of different organelles in the cells and realizing fluorescent imaging of the organelles.
The working concentration of the fluorescent probe in the medium (Dulbecco's modified medium, DMEM) is 0.1. Mu. Mol/L to 1. Mu. Mol/L, preferably 1. Mu. Mol/L to 100. Mu. Mol/L.
In some embodiments, the ultraviolet light wavelength is 200nm to 500nm, preferably 350nm to 450nm.
The azabicyclo-naphthalene fluorescent molecular probe provided by the invention has the characteristics of DSE or AIE luminescence, and has the advantages of narrow half-width, large Stokes shift, good light stability and high fluorescence intensity. In addition, the synthesis method of the azadicyclopentanaphthalene derivative is simple and convenient to operate. Meanwhile, the fluorescent probe molecules can be specifically combined with different organelles in cells, and the labeling and fluorescent imaging of the specific organelles are realized.
Drawings
FIG. 1 is a graph showing fluorescence properties of aggregation-induced fluorescence probe I-1 in dimethyl sulfoxide/water solutions of different ratios.
FIG. 2 is a graph showing fluorescence spectra of aggregation-induced fluorescence probe I-2 in different ratios of dimethyl sulfoxide/water solutions.
FIG. 3 is a graph showing fluorescence spectra of aggregation-induced fluorescence probe I-3 in dimethyl sulfoxide/water solutions of different ratios.
FIG. 4 is a graph showing fluorescence spectra of aggregation-induced fluorescence probe I-4 in different ratios of dimethyl sulfoxide/water solutions.
FIG. 5 is a fluorescence imaging of fluorescent probes I-1, I-2, I-3, I-4 in a cell for labeling different organelles.
Detailed Description
The technical solutions of the present invention will be clearly and completely described with reference to the drawings and examples, and the specific examples described herein are only for explaining the present invention and are not intended to limit the present invention.
Example 1: the compound 4, 11-bis (4- (tert-butyl) phenyl) imidazo [1',2':1,6] pyrido [3,4-e ]
Preparation of imidazo [1,2-a ] pyrazine-6-carbonitrile (I-1).
Step a): imidazole (0.68 g,0.01 mol) was dissolved in 4 mL of anhydrous pyridine under nitrogen protection, and triethylamine (4.2 mL,0.03 mol) was added, the reaction mixture was cooled with an ice bath, and 4-tert-butylbenzoyl chloride (5.88 g,0.03 mol) was added dropwise under the ice bath, then the mixture was stirred at room temperature for 2 hours, after the completion of the reaction of the starting materials was monitored by thin layer chromatography, aqueous NaOH (40 w/w%,2 mL) was carefully added, and the mixture was refluxed at 100 ℃ for 1 hour, then the reaction mixture was poured into cold water (20 mL) and stirred for 1 hour, and filtered to obtain a solid, which was rinsed three times with 20mL of water and dried in an oven at 70 ℃ for 12 hours, to obtain an off-white intermediate M1.
Step b): intermediate M1 (0.912 g,4.0 mmol) obtained in step a) above was dissolved in 10 ml of ethyl acetateCesium carbonate (1.564 g,4.8 mmol) was added to a mixed solvent of nitrile and 10 ml of methylene chloride at room temperature and stirred for 15 minutes, bromoacetonitrile (0.571 g,4.8 mmol) was then added dropwise, the mixture was stirred at room temperature for 2 hours again, after completion of the reaction of the starting materials by thin layer chromatography, cesium carbonate was removed by filtration, and the obtained liquid was concentrated under reduced pressure, and the obtained off-white solid was recrystallized in 15 ml of ethanol to obtain white intermediate M2 (0.961 g,90% yieldd); 1 H NMR(500MHz,CDCl 3 ):δ8.27(2H,d,J＝7.0Hz),7.53(2H,d,J＝7.0Hz),7.33(1H,d,J＝0.5Hz),7.32(1H,d,J＝0.5Hz),5.49(2H,s),1.35(9H,s)； 13 C NMR(125MHz,CDCl 3 ):δ183.74,157.32,142.31,133.61,130.95,130.39,125.37,124.99,114.04,36.48,35.20,31.06；HRMS(ESI)m/z calcd.for C 16 H 17 N 3 O[M+H] + 268.1450,found 268.1455。
step c): intermediate M2 (0.80 g,3 mmol) was dissolved in 10 ml of anhydrous tetrahydrofuran under nitrogen protection, potassium tert-butoxide (0.67 g,6 mmol) was added at room temperature, the mixture was stirred at room temperature for 30 minutes, after the completion of the reaction of the starting materials was monitored by thin layer chromatography, the reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give yellow solid I-1 (0.530 g, 71%); 1 H NMR(500MHz,CDCl 3 ):δ8.75(2H,dt,J＝8.5,2.0Hz),8.20(1H,d,J＝0.5Hz),8.00(1H,d,J＝1.0Hz),7.71(2H,d,J＝8.5Hz),7.61(2H,dt,J＝7.0,1.5Hz),7.48─7.46(2H,m),6.87(1H,d,J＝1.0Hz),1.46(3H,s),1.40(3H,s)； 13 C NMR(125MHz,CDCl 3 ):δ155.70,153.89,153.61,143.54,137.86,137.78,133.86,133.07,131.87,130.41,129.07,128.97,127.09,125.63,122.75,121.45,118.34,113.22,112.29,107.55,35.10,35.07,31.39,31.18；HRMS(ESI)m/z calcd.for C 32 H 30 N 6 [M+H] + 499.2610,found 499.2615。
example 2: preparation of 4, 11-bis (4- (tert-butyl) phenyl) -6-cyano-5-octadecylimidazo [1',2':1,6] pyrido [3,4-e ] imidazo [1,2-a ] pyrazin-5-ium iodide (I-2).
Step d): compound I-1 (50 mg,0.1 mmol) and 1-iodooctadecane (77 mg,0.2 mmol) were dissolved in 5 ml acetonitrile, the mixture was placed in a pressure-resistant tube and stirred at 120 ℃ for reaction for 12 hours, after monitoring the completion of the reaction of the starting materials by thin layer chromatography, the reaction mixture was concentrated under reduced pressure, 5 ml of ethyl acetate was added to the obtained residue and sonicated for 30 minutes, and the obtained solid was filtered, and rinsed twice with 5 ml of ethyl acetate to obtain yellow solid I-2 (86 mg, 86%); 1 H NMR(500MHz,CDCl 3 ):δ8.74(2H,d,J＝8.5Hz),8.44(2H,d,J＝8.0Hz),7.88(2H,d,J＝8.0Hz),7.75(2H,d,J＝8.0Hz),7.61(1H,s),7.60(1H,s),7.48(1H,s),6.46(1H,s),3.72(2H,t,J＝8.0Hz),1.64─1.60(2H,m),1.45(9H,s),1.38(9H,s),1.25─1.10(28H,m),0.88─0.85(5H,m)； 13 C NMR(125MHz,CDCl 3 ):δ156.78,156.29,156.10,138.13,136.04,135.45,134.28,131.12,130.98,130.64,130.15,130.12,127.67,125.77,125.53,118.62,117.78,114.38,110.79,110.53,50.88,35.36,35.21,31.94,31.33,31.11,31.01,29.73,29.71,29.68,29.51,29.38,28.94,26.41,22.71,14.15；HRMS(ESI)m/z calcd.for C 50 H 67 IN 6 [M-I] + 751.5422,found 751.5431。
example 3: preparation of 4, 11-bis (4- (tert-butyl) phenyl) -6-cyano-5- (2-morpholinoethyl) imidazo [1',2':1,6] pyrido [3,4-e ] imidazo [1,2-a ] pyrazin-5-ium iodide (I-3).
Step d): dissolving compound I-1 (50 mg,0.1 mmol) and (4-iodobutyl) triphenyl phosphonium iodide (114 mg,0.2 mmol) in 5 ml acetonitrile, placing the mixture in a pressure-resistant tube, stirring and reacting for 12 hours at 120 ℃, concentrating the reaction mixture under reduced pressure after the reaction of the raw materials is monitored by thin layer chromatography, adding 5 ml of ethyl acetate into the obtained residue, and carrying out ultrasonic treatment for 30 minutes, filtering, and leaching the obtained solid twice with 5 ml of ethyl acetate to obtain yellow solid I-3 (65 mg, 61%); 1 H NMR(500MHz,CDCl 3 ):δ9.14(1H,d,J＝1.0Hz),8.73(2H,d,J＝8.5Hz),8.00(1H,d,J＝1.5Hz),7.96(2H,d,J＝8.5Hz),7.78─7.67(17H,m),7.59(2H,d,J＝9.0Hz),7.48(1H,d,J＝1.5Hz),6.50(1H,d,J＝1.5Hz),4.11(2H,t,J＝6.0Hz),3.41─3.35(2H,m),2.06─2.01(2H,m),1.82─1.76(2H,m),1.36(9H,s),1.36(9H,s)； 13 C NMR(125MHz,CDCl 3 ):δ156.68,156.22,156.16,138.17,136.45,135.65,135.14,135.12,134.28,134.05,133.97,133.82,133.74,131.60,131.20,130.98,130.72,130.62,130.51,130.31,130.25,127.63,125.71,125.40,118.58,117.96,117.87,117.19,112.59,110.81,110.53,49.39,35.32,35.19,31.37,31.11,29.69,19.50,19.48；HRMS(ESI)m/z calcd.for C 54 H 53 I 2 N 6 P[M-2I] 2+ 408.2029,found 408.2030。
example 4: preparation of 4, 11-bis (4- (tert-butyl) phenyl) -6-cyano-5- (4- (triphenylphosphine) butyl) imidazo [1',2':1,6] pyrido [3,4-e ] imidazo [1,2-a ] pyrazine-5-iodide (I-4).
Step d): compound I-1 (50 mg,0.1 mmol) and 4- (2-iodoethyl) morpholine (48 mg,0.2 mmol) were dissolved in 5 ml of acetonitrile, the mixture was placed in a pressure-resistant tube and stirred at 120 ℃ for 12 hours, after monitoring the completion of the reaction of the starting materials by thin layer chromatography, the reaction mixture was concentrated under reduced pressure, 5 ml of ethyl acetate was added to the obtained residue and sonicated for 30 minutes, and the obtained solid was filtered, and the obtained solid was rinsed twice with 5 ml of ethyl acetate to obtain yellow solid I-4 (39 mg, 53%); 1 H NMR(500MHz,CDCl 3 ):δ8.75(2H,d,J＝8.5Hz),8.53(1H,d,J＝2.0Hz),8.35(1H,d,J＝2.0Hz),7.90(2H,d,J＝8.5Hz),7.77(2H,d,J＝8.5Hz),7.62(2H,d,J＝8.5Hz),7.50(1H,d,J＝1.0Hz),6.42(1H,d,J＝1.5Hz),3.85(2H,t,J＝5.5Hz),3.62(4H,t,J＝4.5Hz),2.54(2H,t,J＝5.5Hz),2.41─2.39(4H,m),1.46(9H,s),1.39(9H,s)； 13 C NMR(125MHz,CDCl 3 ):δ156.94,156.60,156.26,138.16,136.21,135.46,134.39,131.34,131.06,131.01,130.27,130.12,127.90,125.83,125.58,118.57,117.70,113.47,110.76,110.47,66.86,57.17,53.32,47.26,35.42,35.24,31.35,31.11；HRMS(ESI)m/z calcd.for C 38 H 42 IN 7 [M-I] + 612.3445,found 612.3452。
example 5: characterization of fluorescence properties in the aggregated state of each fluorescent probe.
The fluorescent probes I-1, I-2, I-3 and I-4 of example 1, example 2, example 3 and example 4 were dissolved in dimethyl sulfoxide, respectively, to prepare 1mmol/L stock solutions, 40. Mu.L of the stock solutions were added to a mixture of 3800. Mu.L of water and 160. Mu.L of dimethyl sulfoxide, and the concentration of the mixed solution was 10. Mu.mol/L of dimethyl sulfoxide/water containing 95% of water, and the fluorescent properties of each fluorescent probe in an aggregated state were measured.
Table 1: fluorescent Properties of Compounds I-1, I-2, I-3 and I-4
Compounds of formula (I) Maximum excitation wavelength Maximum emission wavelength Stokes shift Quantum yield Half width of peak
I-1 395 556 161 7.5％ 70
I-2 374 526 152 15.4％ 44
I-3 372 529 157 16.1％ 43
I-4 369 525 156 11.2％ 46
Experimental results show that the compounds I-1, I-2, I-3 and I-4 prepared in the invention can emit fluorescence with different wavelengths under the excitation of ultraviolet light with different wavelengths under the aggregate state. The maximum emission wavelength is 556 nm, 526 nm, 529 nm and 525nm, and the Stokes shift of each probe is about 150nm, and has large Stokes shift. Meanwhile, the quantum yield of each fluorescent probe in the aggregation state is 7.5%, 15.4%, 16.1% and 11.2%, respectively, and the fluorescent probe has higher quantum yield. In addition, the half-widths of the fluorescent probes I-2, I-3 and I-4 are less than 50nm, and have extremely narrow fluorescence emission peaks. The fluorescence property characterization experiments prove that the probes have good fluorescence emission properties and can be used as fluorescent probes for biological imaging.
Example 6: detection of DSE properties of fluorescent probe I-1.
A stock solution of 1mmol/L of the I-1 fluorescent probe prepared in example 5 was added to a 5 ml centrifuge tube to prepare 4 ml of dimethyl sulfoxide/water solutions of different water contents having a concentration of 10. Mu. Mol/L, wherein the ratio of water was 0%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, and the fluorescence spectra of the respective solutions were measured, and the results are shown in FIG. 1.
When the proportion of water in the solution was 0%, the maximum emission wavelength was 550nm, and as the proportion of water in the solution was gradually increased from 0% to 60%, the fluorescent probe I-1 began to aggregate in the solution and showed a gradual decrease in fluorescence intensity, indicating that the fluorescent probe exhibited aggregation-induced quenching properties. However, a further increase in the water ratio did not significantly affect the fluorescence emission intensity, and the fluorescence emission intensity of the solution with a water ratio of 95% was 0.35 times that of the solution with a water ratio of 0%, indicating that the fluorescent probe I-1 still had strong fluorescence emission in the aggregated state. Furthermore, as shown in FIG. 1, the solution, solid and crystal of fluorescent probe I-1 all exhibited bright fluorescence emission, which is consistent with its DSE properties.
Example 7: detection of AIE properties of fluorescent probes I-2, I-3 and I-4.
1mmol/L stock solutions of the I-2, I-3 and I-4 fluorescent probes prepared in example 5 were taken and respectively added into 5 ml centrifuge tubes to prepare 4 ml of dimethyl sulfoxide/water solutions with different water contents and concentration of 10 mu mol/L, wherein the water ratios were respectively 0%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 95%, and the fluorescence properties of the solutions were respectively tested, and the fluorescence spectra thereof are respectively shown in FIG. 2, FIG. 3 and FIG. 4.
When the proportion of water in the solution was 0%, the maximum emission wavelength was around 520nm, and as the proportion of water in the solution was gradually increased, the fluorescence probes I-2, I-3 and I-4 began to aggregate in the solution, and the fluorescence peak intensities thereof were also gradually increased, indicating that these fluorescence probes all exhibited aggregation-induced fluorescence properties.
Example 8: specific organelle imaging detection in cells.
(1) Detection of lipid droplets in fluorescent probe I-1 specific labeled cells
HeLa cells were grown at 1X 10 cells per well 5 Is inoculated on 24-well culture plates using Dulbecco's modified medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 0.1mg/mL streptomycin and 100 units/mL penicillin at 37℃and 5% CO 2 The environment was allowed to adhere overnight. After pretreatment with oleic acid at a final concentration of 220. Mu.g/mL for 4 hours at 37℃the cells were treated with fluorescent probe I-1 at a final concentration of 10. Mu.M. The cells were further incubated at 37℃for 30 min, then washed twice with PBS. Nile red was then added at a final concentration of 5. Mu.M, incubated at 37℃for 30 minutes, washed twice with PBS, and imaged with confocal fluorescence microscopy.
The fluorescence imaging results are shown in FIG. 5, and the imaging results of the fluorescent probe I-1 (excited at 405nm and emitted at 530-580 nm) are highly coincident with the imaging results of the positive control nile red (excited at 550nm and emitted at 580-640 nm), which shows that the fluorescent probe I-1 can specifically label lipid droplets in cells and realize fluorescence imaging.
(2) Detection of cell membrane imaging in fluorescent probe I-2 specific labeled cells
HeLa cells were grown at 1X 10 cells per well 5 Is inoculated on 24-well culture plates using Dulbecco's modified medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 0.1mg/mL streptomycin and 100 units/mL penicillin at 37℃and 5% CO 2 The environment was allowed to adhere overnight. HeLa cells were incubated with fluorescent probes I-2 and DiI (1. Mu.M) at a final concentration of 10. Mu.M for 15 minutes at 37℃and then washed twice with PBS. Imaging was detected with confocal fluorescence microscopy.
The fluorescence imaging results are shown in FIG. 5, and the imaging results of the fluorescent probe I-2 (excited at 405nm and emitted at 500-540 nm) are highly coincident with the imaging results of the positive control DiI (excited at 550nm and emitted at 580-640 nm), which shows that the fluorescent probe I-2 can specifically label the cell membrane in the cell and realize fluorescence imaging.
(3) Mitochondrial imaging detection in fluorescent probe I-3 specific labeled cells
HeLa cells were grown at 1X 10 cells per well 5 Is inoculated on 24-well culture plates using Dulbecco's modified medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 0.1mg/mL streptomycin and 100 units/mL penicillin at 37℃and 5% CO 2 Wall attachment in environmentOvernight. HeLa cells were incubated with fluorescent probe I-3 at a final concentration of 10. Mu.M and Mito-tracker Red (1. Mu.M) for 2 hours at 37℃and then washed twice with PBS. Imaging was detected with confocal fluorescence microscopy.
The fluorescence imaging results are shown in FIG. 5, and the imaging results of the fluorescent probe I-3 (excited at 405nm and emitted at 500-540 nm) are highly coincident with the imaging results of the positive control Mito-tracker Red (excited at 550nm and emitted at 580-640 nm), which indicates that the fluorescent probe I-3 can specifically label mitochondria in cells and realize fluorescence imaging.
(4) Lysosome imaging detection in fluorescent probe I-4 specific labeled cells
HeLa cells were grown at 1X 10 cells per well 5 Is inoculated on 24-well culture plates using Dulbecco's modified medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 0.1mg/mL streptomycin and 100 units/mL penicillin at 37℃and 5% CO 2 The environment was allowed to adhere overnight. HeLa cells were incubated with fluorescent probe I-4 at a final concentration of 10. Mu.M and Lyso-tracker Red (1. Mu.M) for 1 hour at 37℃and then washed twice with PBS. Imaging was detected with confocal fluorescence microscopy.
The fluorescence imaging results are shown in FIG. 5, and the imaging results of the fluorescent probe I-4 (excited at 405nm and emitted at 500-540 nm) are highly coincident with the imaging results of the positive control Lyso-tracker Red (excited at 550nm and emitted at 580-640 nm), which indicates that the fluorescent probe I-4 can specifically label lysosomes in cells and realize fluorescence imaging.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.

Claims (10)

1. The azabicyclo-naphthalene fluorescent molecular probe is characterized in that the azabicyclo-naphthalene derivative has the following structure:
2. the method for preparing the azadicyclopentane fluorescent molecular probe according to claim 1, which is characterized by comprising the following steps:
a) In pyridine, 4-tertiary butyl benzoyl chloride and imidazole generate M1 under the catalysis of triethylamine;
b) In a mixed solvent of acetonitrile and dichloromethane, the intermediate M1 reacts with bromoacetonitrile under the catalysis of cesium carbonate to generate an intermediate M2;
c) In tetrahydrofuran, the intermediate M2 generates a compound I-1 under the catalysis of potassium tert-butoxide;
d) In acetonitrile, the compound I-1 reacts with different iodides to respectively obtain compounds I-2, I-3 and I-4;
the synthetic route is as follows:
3. the preparation method according to claim 2, wherein in step a), imidazole is dissolved in anhydrous pyridine under nitrogen protection and triethylamine is added, the reaction mixture is cooled with ice bath and 4-tert-butylbenzoyl chloride is added dropwise under ice bath, then the mixture is stirred at room temperature for 2 hours, naOH aqueous solution is carefully added and the mixture is refluxed for 1 hour, the reaction mixture is poured into cold water and stirred for 1 hour, the solid is filtered, washed three times with 20ml of water and dried at 70 ℃ to obtain the yellowish white intermediate M1.
4. The preparation method according to claim 2, wherein in step b), the intermediate M1 is dissolved in a mixed solvent of acetonitrile and methylene chloride, cesium carbonate is added at room temperature and reacted with stirring for 15 minutes, bromoacetonitrile is then added dropwise, the mixture is reacted with stirring for 2 hours at room temperature, the reaction solution is filtered and the obtained liquid is concentrated under reduced pressure, and the obtained residue is recrystallized in ethanol to obtain the intermediate M2.
5. The preparation method according to claim 2, wherein in step c), the intermediate M2 is dissolved in anhydrous tetrahydrofuran under the protection of nitrogen, potassium tert-butoxide is added at room temperature, the reaction mixture is stirred at room temperature for 30 minutes, then the reaction solution is concentrated under reduced pressure, and the residue is purified by silica gel column chromatography to obtain yellow solid I-1.
6. The process according to claim 2, wherein in step d), the compounds I-1 and 1-iodooctadecane, (4-iodobutyl) triphenylphosphonium iodide or 4- (2-iodoethyl) morpholine are dissolved in acetonitrile, the mixture is placed in a pressure-resistant tube and reacted for 12 hours under stirring at 120 ℃, the reaction mixture is concentrated under reduced pressure, ethyl acetate is added to the residue and sonicated for 30 minutes, and the resulting solid is filtered, and the obtained solid is rinsed twice with ethyl acetate to obtain yellow solids I-2, I-3 and I-4, respectively.
7. Use of an azadicyclopentane fluorescent molecular probe according to claim 1 for fluorescent labelling and imaging of organelles, wherein the organelles are cell membranes, lipid droplets, mitochondria and lysosomes.
8. The use according to claim 7, wherein fluorescent probe I-1 is capable of labelling lipid droplets in cells, fluorescent probe I-2 labels cell membranes in cells, fluorescent probe I-3 labels mitochondria in cells, and fluorescent probe I-4 labels lysosomes in cells.
9. The use according to claim 8, wherein the fluorescent molecular probes are added directly or dissolved in a solution to a medium containing cells, the fluorescent probes are taken up by the cells and aggregated at the corresponding organelles, and fluoresce under uv-visible light excitation, thereby completing the labeling and imaging of the organelles in the cells.
10. The use according to claim 8, wherein the working concentration of the fluorescent probe is 0.1 μmol/L to 1mol/L; the wavelength of the ultraviolet visible light is 200nm to 500nm.
CN202311025550.4A 2023-08-15 2023-08-15 Azabicyclo-naphthalene fluorescent molecular probe and preparation and application thereof Pending CN117088881A (en)

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