Preparation method and application of near-infrared emission fluorescent probe

Abstract

The invention discloses a preparation method and application of a near-infrared emission fluorescent probe. The invention discloses the near-infrared emission fluorescent probe, a preparation process of the near-infrared emission fluorescent probe and application of the near-infrared emission fluorescent probe to cell lysosome imaging. The fluorescent probe is located in a near-infrared region, the maximum absorption peak is located at 660-700 nm, the maximum emission peak is located at 800-810 nm, and the fluorescent probe has deep tissue penetrating power, can specifically target cell lysosome, has good structural stability, is very low in toxicity to cells, can be used for long-acting tracing of the lysosome, and can be used for detecting the cell lysosome. And important support can be provided for scientists to deeply understand the function of lysosomes and the effect of lysosomes in diseases and develop a new treatment strategy.

Description

Technical Field

[0001] The invention belongs to the field of fluorescent biotechnology materials, and specifically relates to the preparation and application of a cell lysosome-targeted near-infrared fluorescent probe. Background Art

[0002] Currently, common molecular imaging technologies such as computed tomography (CT), X-ray, ultrasound (US) and magnetic resonance imaging (MRI) are widely used in medical diagnosis, but their limitations in spatial resolution and dynamic monitoring have prompted scientists to continuously explore new imaging technologies and methods. Fluorescence imaging is one of the fastest-growing and most widely used imaging technologies in the field of biomedical research, providing a method for diagnosing and surgically treating diseases in organisms. Near-infrared organic fluorescent probes are compounds that emit fluorescence in the near-infrared spectral region (usually in the wavelength range of 700-1100 nanometers). This type of probe has a wide range of applications in biomedical imaging, chemical detection, materials science and other fields. Due to the strong penetration of near-infrared light into biological tissues, near-infrared organic fluorescent probes are particularly suitable for imaging in vivo or in cells, which can reduce damage to samples and improve detection sensitivity.

[0003] Lysosome is an indispensable organelle in eukaryotic cells, a degradation center and metabolic signal center of living cells, and is involved in important physiological processes such as autophagy, cell death and cell aging. Fluorescent probes have been widely used in molecular markers and organelle tracing due to their advantages such as strong specificity and high biocompatibility. Lysosomal fluorescent probes have broad application prospects in the fields of cell biology, pathology and pharmacology. However, the lysosomal fluorescent probes currently developed are mostly emitted in the visible light region, and have poor penetration ability, which limits their application in deep tissue imaging. Many lysosomal targeting probes rely on capturing positively charged fat-soluble amines, and these ligands may be toxic to living cells, affecting long-term tracking. In contrast, the fluorescent probe of the present invention can be specifically located in the cell lysosome, and is located in the near-infrared region, has a deeper tissue penetration ability, higher biocompatibility and good structural stability, and can provide important support for scientists to more deeply understand the function of lysosomes and their role in diseases, and to develop new treatment strategies. Summary of the invention

[0004] The purpose of the present invention is to provide a type of organic small molecule fluorescent probe with near-infrared emission, high structural stability and good biocompatibility, which has a cell lysosome targeting effect and a preparation method thereof.

[0005] The near-infrared emitting organic small molecule fluorescent probe provided by the present invention has a general structural formula (1) as shown below: (1) Among them, R 1H, CH 3 , CN or other groups or halogen atoms. 2 It is hexanoic acid group or alkyl chain of different lengths.

[0006] The preparation method of the near-infrared fluorescent probe proposed in the present invention has a chemical synthesis route as follows:

[0007] Among them, R 1 H, CH 3 , CN or other groups or halogen atoms. 2 It is hexanoic acid group or alkyl chain of different lengths.

[0008] The specific steps of preparation are: (1) Synthesis of intermediate 1 Under nitrogen protection, with toluene as solvent, (2-(4-bromophenyl)ethylene-1,1,2-triyl)triphenyl, tri(dibenzylideneacetone)dipalladium(0) and tri-tert-butylphosphine tetrafluoroborate were added after stirring at room temperature for 10 minutes, and then sodium tert-butoxide and aniline were added, and the mixture was stirred and refluxed at 120 °C for 24 h. The solvent was evaporated and the crude product was purified by silica gel chromatography to prepare intermediate 1; (2) Synthesis of intermediate 2 Under nitrogen protection, with toluene as solvent, intermediate 1, tri(dibenzylideneacetone)dipalladium(0), tri-tert-butylphosphine tetrafluoroborate, sodium tert-butoxide and 5-bromo-2,2'-bithiophene-5'-carboxaldehyde were stirred and refluxed at 100 °C for 48 h. The solvent was evaporated and the crude product was purified by silica gel chromatography to prepare intermediate 2; (3) Synthesis of near-infrared fluorescent probes Under nitrogen protection, with acetic acid and acetic anhydride as solvents, intermediate 2, compound n and triethylamine were stirred and refluxed at 70°C for 5 h. The solvent was evaporated and the crude product was purified by silica gel chromatography to prepare a near-infrared fluorescent probe.

[0009] Compared with the prior art, the invention has the following beneficial effects.

[0010] The near-infrared fluorescent probe of the present invention has the following characteristics: (1) The fluorescent probe of the present invention emits fluorescence in the near-infrared region, with a maximum absorption peak at 660-700 nm and a maximum emission peak at 800-810 nm; (2) The fluorescent probe of the present invention has good structural stability and can specifically target cell lysosomes; (3) The fluorescent probe of the present invention has very low toxicity to cells and can be used for long-term tracing of lysosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the UV absorption spectrum of the near-infrared fluorescent probe (10 µM) in ethanol.

[0012] Figure 2 This is the fluorescence emission spectrum of the near-infrared fluorescent probe (10 µM) in ethanol.

[0013] Figure 3 Confocal microscopy images of the colocalization of near-infrared fluorescent probe and Mito-Tracker Green in A549 cells.

[0014] Figure 4 Confocal microscopy image of the colocalization of the near-infrared fluorescent probe and Lyso-Tracker Green in A549 cells.

[0015] Figure 5 The figure shows the cytotoxicity of A549 cells co-incubated with near-infrared fluorescent probe. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described with the following specific embodiments, but the present invention is by no means limited to these examples. The following description is only a preferred embodiment of the present invention, which is only used to explain the present invention and cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that any modification, substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0017] The photosensitizer of the present invention and the specific implementation of its preparation method and application are further described below in conjunction with the accompanying drawings and examples.

[0018] Embodiment 1: Preparation of near-infrared fluorescent dye 1a, the compound structure is as follows: 1a The specific synthetic route is as follows:

[0019] The synthesis method is as follows: Specific steps: Place the magneton, compound 1 (0.216 g, 0.610 mol) and intermediate 2 (0.25 g, 0.406 mol) into a 100 mL two-necked flask, assemble a reflux device, replace with vacuum-nitrogen three times, add 2 mL of acetic acid, 0.2 mL of acetic anhydride and 0.2 mL of triethylamine with a syringe, heat to 70°C and reflux for 5 hours. Evaporate the solvent with an oil pump, separate the crude product with a silica gel column, and purify with CH 2 Cl 2 :CH 3 The fluorescent probe 1a was obtained by elution with OH=30:1 eluent. 1H NMR (400 MHz, Chloroform-d) δ 8.52 (d, J = 15.0 Hz, 1H), 8.34 (s, 1H), 7.49 (d, J = 15.3Hz, 2H), 7.34-7.29 (m, 3H), 7.18 (d, J = 7.5 Hz, 3H), 7.15-7.11 (m, 6H), 7.09(s, 5H), 7.06-7.01 (m, 4H), 6.94 (q, J = 8.6 Hz, 5H), 6.43 (d, J = 4.1 Hz,1H), 4.53 (s, 2H), 3.14 (q, J = 7.3 Hz, 2H), 2.39 (s, 1H), 2.04 (s, 1H), 1.93(s, 2H), 1.82 (s, 5H), 1.73 (s, 2H), 1.57 (s, 2H), 1.39 (t, J = 7.3 Hz, 4H).HRMS (ESI) m / z: [M] + , calculated, 871.3386; actual value, 871.33884.

[0020] Embodiment 2: Preparation of near-infrared fluorescent dye 1b, the compound structure is as follows: 1b The specific synthetic route is as follows:

[0021] The synthesis method is as follows: Specific steps: Place the magneton, compound 2 (0.13 g, 0.325 mol) and intermediate 2 (0.2 g, 0.325 mol) into a 100 mL two-necked flask, assemble a reflux device, replace with vacuum-nitrogen three times, add 2 mL of acetic acid, 0.2 mL of acetic anhydride and 0.2 mL of triethylamine with a syringe, heat to 70°C and reflux for 5 hours. Evaporate the solvent with an oil pump, separate the crude product with a silica gel column, and purify with CH 2 Cl 2 :CH 3 The fluorescent probe 1b was obtained by elution with OH=35:1 eluent. 1H NMR (400 MHz, Methanol-d4) δ 8.48 (d, J = 14.8 Hz, 1H), 7.82 (s, 1H), 7.69 (s, 1H), 7.58 (d, J = 8.1Hz, 1H), 7.48 (d, J = 8.6 Hz, 1H), 7.34 (s, 1H), 7.26 (d, J = 7.9 Hz, 2H), 7.19 (s, 2H), 7.07 (d, J = 11.9 Hz, 6H), 6.99 (d, J = 9.2 Hz, 4H), 6.92 (d, J= 11.6 Hz, 4H), 6.84 (d, J = 15.9 Hz, 5H), 6.35 (s, HRMS (ESI) m / z:[M] + , calculated, 905.2997; actual value, 905.29981.

[0022] Embodiment 3: Preparation of near-infrared fluorescent dye 1c, the compound structure is as follows: 1c The specific synthetic route is as follows:

[0023] The synthesis method is as follows: Specific steps: Place the magneton, compound 3 (0.64 g, 2 mol) and intermediate 2 (0.8 g, 1.3 mol) into a 100 mL two-necked flask, assemble a reflux device, replace with vacuum-nitrogen three times, add 2 mL of acetic acid, 0.2 mL of acetic anhydride and 0.2 mL of triethylamine with a syringe, heat to 70°C and reflux for 5 hours. Evaporate the solvent with an oil pump, separate the crude product with a silica gel column, and purify with CH 2 Cl 2 :CH 3 The fluorescent dye 1c was obtained by elution with OH=40:1 eluent. 1H NMR (400 MHz, Methanol-d4) δ8.62-8.51 (m, 1H), 7.88 (d, J = 7.1 Hz, 1H), 7.69 (s, 2H), 7.57 (s, 3H), 7.35(s, 3H), 7.15 (s, 6H), 7.08 (s, 5H), 6.99 (s, 4H), 6.94 (d, J = 9.9 Hz, 5H), 6.44 (d, J = 9.2 Hz, 1H), 4.48 (s, 2H), 1.91 (s, 2H), 1.80 (d, J = 9.3 Hz, 6H), 1.43 (s, 6H), 1.22 (s, 15H), 0.86-0.80 (m, 3H). HRMS (ESI) m / z: [M] + , calculated, 925.4584; actual value, 925.45085.

[0024] Embodiment 4: UV absorption spectrum: Prepare 10 µM ethanol solutions of 1a, 1b and 1c respectively and measure the UV absorption spectra. The results are as follows: Figure 1 .

[0025] Embodiment 5: Fluorescence emission spectra: 10 µM ethanol solutions of 1a, 1b and 1c were prepared respectively and the fluorescence emission spectra (Ex = 660 nm) were measured. The results are shown in Figure 2 .

[0026] Embodiment 6: Mitochondrial colocalization fluorescence confocal laser scanning microscopy imaging: A549 cells were seeded in a 35 mm glass bottom culture dish (special for confocal microscope) at a density of 3*10 5 / dish, and culture overnight. Add 10 µM of probes 1a, 1b, and 1c and culture for 24 hours, then add 100 nM Mito-tracker Green and continue to culture for half an hour. Aspirate the culture medium and wash with PBS three times. Record cell fluorescence with a confocal microscope. The results are as follows Figure 3 .

[0027] Embodiment 7: Lysosome colocalization fluorescence confocal laser scanning microscopy imaging: A549 cells were seeded in a 35 mm glass bottom culture dish (special for confocal microscope) at a density of 3*10 5 / dish, and culture overnight. Add 10 µM of probes 1a, 1b, and 1c and culture for 24 hours, then add 100 nM Lyso-tracker Green and continue to culture for half an hour. Aspirate the culture medium and wash with PBS three times. Record cell fluorescence with a confocal microscope. The results are as follows Figure 4 .

[0028] Embodiment 8: Cytotoxicity evaluation: A549 cells were seeded in a 96-well plate at a density of 20,000 cells / well and cultured for 24 hours. The original culture medium in the plate was discarded, and probes 1a, 1b and 1c with different concentration gradients were added to each well. Five replicates were set up in each well and cultured for 24 hours. 20 µL of MTT solution was added, incubated for 4 hours, the mixed solution was aspirated, 150 µL of DMSO was added to each well, and shaken at 37 °C for 10 minutes. The ultraviolet absorption value at 570 nm was recorded using an enzyme reader, and the cell growth viability was calculated. Viability (%) = (average absorbance value of the experimental group / average absorbance value of the control group) * 100%, where the control group was cells without drug addition, and the results were as follows. Figure 5 .

Claims

1. A near-infrared fluorescent probe targeting lysosomes, the general structural formula (1) of which is shown below: （1） in, R1 is a group such as H, CH3, CN or a halogen atom. R2 is a caproic acid group or an alkyl chain of different lengths.

2. A method for preparing a fluorescent probe as claimed in claim 1, characterized in that: The synthetic route is as follows: Wherein, R1 is a group such as H, CH3, CN or a halogen atom. R2 is a caproic acid group or an alkyl chain of different lengths.

3. The fluorescent probe as claimed in claim 1, wherein the fluorescence emission is in the near-infrared region, the maximum ultraviolet absorption peak is located at 660 ~ 700 nm, and the maximum fluorescence emission peak is located at 800 ~ 810 nm.

4. The fluorescent probe as claimed in claim 1 has good structural stability and can specifically target cell lysosomes.

5. The fluorescent probe as claimed in claim 1 has very low toxicity to cells and can be used for long-term tracing of lysosomes.

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