CN109535098B - Near-infrared fluorescent probe for viscosity detection and preparation and application thereof - Google Patents
Near-infrared fluorescent probe for viscosity detection and preparation and application thereof Download PDFInfo
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- 229940125904 compound 1 Drugs 0.000 claims description 21
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Abstract
The invention relates to a near-infrared fluorescent probe for viscosity detection and preparation and application thereof, belonging to the field of analysis and detection. The invention has the advantages of cheap and easily obtained raw materials, simple and convenient operation, mild reaction conditions and low production cost, is convenient for industrialization, has larger Stokes displacement, can be used for detecting the near infrared probe of viscosity, and carries out accurate structural characterization on the target compound by means of characterization means such as mass spectrum, nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum. The probe can be applied to detection and diagnosis of human health/diseases, and the change of cell viscosity can be detected by using the probe, so that early warning of diseases can be achieved. The probe has better chemical stability, biocompatibility and selectivity. Laser confocal imaging experiments show that the probe has good cell permeability, has no toxic or side effect on cells and organisms, can realize the detection of the horizontal viscosity of the cells and indicate the viscosity change condition, and can be further applied to the research of early detection of diseases.
Description
Technical Field
The invention relates to the field of organic fluorescent probes, in particular to design of a near-infrared fluorescent probe for viscosity detection and application of the near-infrared fluorescent probe in the field of human health disease detection.
Background
Fluorescent probes are a great discovery in the technical field of chemical sensing in the eighties of the last century, and more fluorescent probes are applied to real-time detection at the molecular level at present. The fluorescence detection technology has high sensitivity, simple operation, strong visibility and small damage to cells and organisms, so that detection objects of the molecular fluorescence probe which is an indispensable detection tool in the fields of clinical analysis, environmental monitoring, biological analysis, life science and the like comprise various ions, small molecules, free radicals, polypeptides and enzymes, and even temperature, polarity, viscosity and the like. When the probe with low molecular weight interacts with the target molecule, the target is often detected by chemical reaction, electrostatic interaction, indirect acting force and other ways. People can use instruments such as a fluorescence microscope, a fluorescence spectrometer, a flow cytometer, a fluorescence living body imaging system and the like to acquire relevant information detected by a fluorescence probe, can detect the concentration of molecules or ions in living cells and the change process of a biological macromolecular structure in real time by means of a fluorescence imaging technology, can also acquire relevant information about the physiological metabolic process of biological tissues, and can also realize fluorescence imaging of a biological living body.
Both the diagnosis and treatment of various diseases and the study of the pathogenesis of the diseases require highly sensitive and specific detection means. On the basis that fluorescence detection, particularly organic small molecule fluorescent probes, have the advantages of high sensitivity and simplicity and convenience in operation on one hand, and on the other hand, researchers can design and synthesize probe molecules meeting specific requirements according to needs, fluorescent probes and fluorescence detection technologies play a great role in the development of life sciences.
In recent years, the development of the subjects of life science, environmental science, material science, life medicine and the like has made higher demands on the performance of the fluorescent probe. Higher sensitivity and selectivity, lower detection limit, higher accuracy and precision, more complete and reliable morphological analysis, faster analysis speed and automation degree, smaller sample size, better biocompatibility, micro-damage or non-damage analysis, living body, real-time analysis, miniaturization and intellectualization of analytical devices and the like. Therefore, the development of fluorescent probes with better performance for biochemical, molecular biological and cell biological research is an urgent and significant task.
As a very important factor in controlling the mass transfer process, viscosity plays a crucial role not only in the chemical-related field but also in the activities between different biological systems in the life science field. The viscosity can control the transfer rate between different substances and the transfer of substances between multi-phase liquids in life activities. For example, intracellular viscosity can affect the transport of nutrients involved in cell life activities and waste products produced by cell metabolism. Abnormal cell microenvironment viscosity has been shown to be associated with a number of diseases such as alzheimer's disease, atherosclerosis, diabetes, and even cellular malignancies. Therefore, the fluorescent probe for detecting viscosity change has great significance in detecting at a cellular molecular level.
Disclosure of Invention
The invention aims to provide a fluorescent probe which has high sensitivity, simple and convenient operation, strong visibility and small damage to cells and organisms, a near-infrared fluorescent probe which has high viscosity sensitivity and prominent near-infrared performance and is used for viscosity detection, and preparation and application thereof. The fluorescent probe can be applied to detection of human health diseases, and can obtain a high-efficiency fluorescence imaging picture.
The technical scheme adopted by the invention is as follows: the preparation and application of a near-infrared probe for detecting viscosity comprise a viscosity-response group unit and a fluorophore with near-infrared performance, and the structure is as follows:
the other technical scheme adopted by the invention is as follows: the preparation method of the near-infrared probe NY3 with the fluorescent probe based on the BD fluorophore comprises the following steps:
2.1 NY 3: mixing compound 1(50-200mg), compound 3(25-100mg), Na2CO3(32.5-130 mg) and a small amount of palladium chloride as a catalyst were mixed in 2-10mL of dioxane and stirred under N2Mixing under protectionThe mixture was stirred at 100 ℃ for 12 h. Then spin-drying, and purifying by silica gel column chromatography to obtain a red solid product NY 3.
Preferably, the molar ratio of the compound 1 to the compound 3 in the preparation method of the fluorescent probe NY3 is 1.2:1-2:1, and the compound 3 is not excessive in the reaction process, otherwise, by-products are generated, and the yield is reduced. And secondly, the reaction must be protected by nitrogen and is in a high-temperature environment, otherwise, other impurities are generated.
Preferably, the preparation method of the near-infrared probe precursor compound 1 for detecting viscosity is as follows:
compound 1: in a 100mL three-necked flask, 6-bromo-N, N-dimethylnaphthalen-2-amine (126.0-504.0mg), 4,4,4',4',5,5,5',5' -octamethyl-2, 2' -bis (1,3, 2-dioxaborolane) (152.35-609.4mg) and CH3COOK (93.15-372.6mg) was added to 2-10mL of a dioxane solution. After addition of the catalyst palladium chloride, the mixture is taken up in N2Stirring at 80 ℃ for 12h under protection. Then, the intermediate product, compound 1, was obtained as a white solid by extraction with EtOAc, spin-drying, and purification by silica gel column chromatography. The reaction formula is as follows:
preferably, in the preparation of intermediate compound 1 in the preparation method 4.1 of the series of two-photon naphthol derivative compounds in the step 4, 6-bromo-N, N-dimethylnaphthalene-2-amine, 4,4,4',4',5,5,5',5' -octamethyl-2, 2' -bis (1,3, 2-dioxaborolane), CH3The molar ratio of COOK is 1:1.2:2-1:2:3, and the time is 12 h.
Preferably, the preparation method of the near-infrared probe precursor compound 3 for detecting viscosity is as follows:
6.1 Compound 3:
(1) compound 2: mixing acetic acid (4.5-18)mL) was heated to 70 ℃ and then NBD-Cl (0.11-0.44 g) and Fe powder (0.09-0.36g) were added. In N2The mixture was stirred at 70 ℃ for 30min with protection. The reaction was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in ethyl acetate and washed with NaHCO3Washing was carried out 3 times. The organic layers were combined and washed with anhydrous Na2SO4And (5) drying. The residue was purified by silica column (ethyl acetate/petroleum ether ═ 1/10) to give compound 2 as a yellow powder, according to the following reaction scheme:
(2) compound 3: to a solution of Compound 2(0.025-0.1g) in acetonitrile (2.5-10mL) was added CH in sequence3I (0.036-0.144mL) and Cs2CO3(150-600 mg). The reaction was then stirred at 85 ℃ for 12 hours. The product was extracted with EtOAc (3X 75 mL). The organic layers were combined and the solvent was evaporated in vacuo. The residue was purified by silica column (petroleum ether/ethyl acetate ═ 20/1) to give compound 3 as an orange-red powder, according to the following reaction scheme:
(1) the mol ratio of the medium NBD-Cl and the Fe powder is 1:3-1:5, acetic acid is heated to 70 ℃ in advance, and then the mixture is stirred for 30min under the protection of nitrogen, otherwise, the yield is greatly reduced.
(2) The intermediate compound 2, CH3I、Cs2CO3The molar ratio of (A) to (B) is 1:4:3-1:5:5, in the reaction process, CH3The molar amount of I should not be too small, otherwise too much by-product is produced, greatly reducing the yield. And the reaction is carried out under the protection of nitrogen gas, otherwise, the reaction yield is extremely low.
Has the advantages that:
the fluorescent probe has the characteristics of good chemical stability, biological compatibility, selectivity and the like. Laser confocal imaging experiments show that the probe has better cell permeability and no toxic or side effect on cells.
The fluorescent probe can be applied to a cell system to detect the change of viscosity, and the mainly used living cell is a HeLa cell strain.
Drawings
1. FIG. 1 shows a synthetic route of viscosity probe NY 3.
2. FIGS. 2-a,2-b,2-c show the hydrogen, carbon and mass spectra data for the viscosity probe NY 3.
3. FIGS. 3-a,3-b,3-c are graphs of the UV absorbance, fluorescence emission and fitted curves of viscosity probe NY3 in solutions of different viscosity sizes (different ratios of methanol to glycerol).
4. FIG. 4 shows the result of the cytotoxic MTT of the viscosity probe NY 3.
5. FIG. 5 is a diagram of the cell image of the viscosity probe NY3 under different viscosity conditions.
Detailed Description
Example 1
The preparation method of the near-infrared viscosity probe NY3 based on the BD (nitrobenzo-2-oxa-1, 3-diazole) fluorophore comprises the following steps:
1.1 Compound 1: in a 100mL three-necked flask, 6-bromo-N, N-dimethylnaphthalen-2-amine (252.0 mg,1.0 mmol), 4,4,4',4',5,5,5',5' -octamethyl-2, 2' -bis (1,3, 2-dioxaborolane) (152.35mg, 0.6mmol) and CH3COOK (186.3mg, 2.0mmol) was added to a 10mL dioxane solution. After addition of the catalyst palladium chloride, the mixture is taken up in N2Stirring at 80 ℃ for 12h under protection. Then, the intermediate product, compound 1, was obtained as a white solid by extraction with EtOAc, spin-drying, and purification by silica gel column chromatography. The reaction formula is as follows:1H NMR(500MHz,DMSO-d6):δ8.23(s,1H),7.76-7.73(m,2H), 7.63(d,J＝8.2Hz 1H),7.15(dd,J1＝2.2,J2＝9.0Hz,1H),6.91(s,1H),3.07(s,6H), 1.39(s,12H).
1.2 Compound 2: acetic acid (9mL) was heated to 70 ℃ and then NBD-Cl (0.11g,0.5mmol) and Fe powder (0.09g, 0.5mmol) were added5 mmol). In N2The mixture was stirred at 70 ℃ for 30min with protection. The reaction was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in ethyl acetate and washed with NaHCO3Washing was carried out 3 times. The organic layers were combined and washed with anhydrous Na2SO4And (5) drying. The residue was purified by silica column (ethyl acetate/petroleum ether ═ 1/10) to give compound 2 as a yellow powder.1H NMR(500MHz,DMSO-d6) δ 7.40(d, J ═ 10Hz,1H),6.75(s,2H),6.28(d, J ═ 10Hz, 1H). The reaction formula is as follows:
1.3 Compound 3: to a solution of compound 2(0.1g,2mmol) in acetonitrile (10mL) was added CH in sequence3I (0.036mL,0.5mmol) and Cs2CO3(600mg,2 mmol). The reaction was then stirred at 85 ℃ for 12 hours. The product was extracted with EtOAc (3X 75 mL). The organic layers were combined and the solvent was evaporated in vacuo. The residue was purified by silica column (petroleum ether/ethyl acetate ═ 20/1) to give compound 3 as an orange-red powder.1H NMR(500MHz,CDCl3) δ 7.19(d, J ═ 8.1Hz,1H),5.94(d, J ═ 8.1Hz,1H), 3.30(s, 6H). The reaction formula is as follows:
1.4 NY 3: mixing compound 1(50mg,0.18mmol), compound 3(100mg,0.6mmol), Na2CO3(130mg,1.2mmol) and a small amount of palladium chloride as a catalyst were mixed in 10mL of dioxane and stirred under N2The mixture was stirred at 100 ℃ for 12h with protection. Then spin-drying, and purifying by silica gel column chromatography to obtain a red solid product NY 3.1H NMR(500MHz,CDCl3):δ1H NMR(500MHz,CDCl3)δ 8.35(s,1H),7.90(dd,J1＝1.5Hz,J2＝8.6,Hz,1H),7.81-7.71(m,3H),7.54(d,J＝ 7.8Hz,1H),7.19(d,J＝7.8Hz,1H),6.21(d,J＝7.8Hz,1H),3.34(s,6H),3.07(s, 6H).13C NMR(125MHz,CDCl3):δ149.97,146.16,138.51,134.22,129.90,129.33, 127.19,127.07,126.96,126.54,126.29,125.54,116.62,108.95,106.28,106.18,42.04, 41.86,40.92,40.90.ESI-MS:Calculated:[M+H]+＝333.17,found:[M+H]+333.13. The reaction formula is as follows:
adding dimethyl sulfoxide solutions of NY3 into a culture dish containing HeLa cells respectively, uniformly mixing the dimethyl sulfoxide solutions with a cell culture solution to ensure that the concentration of NY3 in the culture solution is 10 mu M, and then dividing the culture solution into six groups: 1.37 ℃ group 2.25 ℃ group 3.4 ℃ group 4.4% paraformaldehyde group 5 Nystatin (Nystatin, 10 μ M) group 6 dimethyl sulfoxide (control). 1, 2, and 3 groups are that the probe and the cell are cultured for 0.5h in respective temperature environments (37 ℃, 25 ℃,4 ℃); 4 groups are that after the probe and the cells are cultured for 0.5h at 37 ℃, the cells are fixed by 4 percent paraformaldehyde; group 5 is that after the probe and the cells are cultured for 0.5h at 37 ℃, 10 MuM nystatin culture medium solution is added, and the culture is continued for 0.5 h; group 6 was cultured with DMSO at 37 ℃ for 0.5 h. The plate was then washed 3 times with PBS buffer pH 7.35 and imaged under a confocal microscope (543 nm for single photon excitation and 560-620nm for reception).
Example 2
The preparation method of the near-infrared viscosity probe NY3 based on the BD fluorophore comprises the following steps:
2.1 Compound 1: in a 100mL three-necked flask, 6-bromo-N, N-dimethylnaphthalen-2-amine (100mg, 0.40mmol), 4,4,4'4', 5,5,5',5' -octamethyl-2, 2' -bis (1,3, 2-dioxaborolane) (50mg, 0.20mmol) and CH3COOK (100mg, 1.01mmol) was added to a 5mL dioxane solution. After addition of the catalyst palladium chloride, the mixture is taken up in N2Stirring at 80 ℃ for 12h under protection. Then, the intermediate product, compound 1, was obtained as a white solid by extraction with EtOAc, spin-drying, and purification by silica gel column chromatography.
2.2 Compound 2: acetic acid (5mL) was heated to 70 ℃ and then NBD-Cl (0.22g,1mmol) and Fe powder (g: (R) (R))0.27g,0.15 mmol). In N2The mixture was stirred at 70 ℃ for 30min with protection. The reaction was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in ethyl acetate and washed with NaHCO3Washing was carried out 3 times. The organic layers were combined and washed with anhydrous Na2SO4And (5) drying. The residue was purified by silica column (ethyl acetate/petroleum ether ═ 1/10) to give compound 2 as a yellow powder.
2.3 Compound 3: to a solution of compound 2(0.05g,1mmol) in acetonitrile (5mL) was added CH in sequence3I (0.018mL,0.25mmol) and Cs2CO3(300mg,1 mmol). The reaction was then stirred at 85 ℃ for 12 hours. The product was extracted with EtOAc (3X 75 mL). The organic layers were combined and the solvent was evaporated in vacuo. The residue was purified by silica column (petroleum ether/ethyl acetate ═ 20/1) to give compound 3 as an orange-red powder.
2.4 NY 3: mixing Compound 1(150mg,0.54mmol), Compound 3(50mg,0.3mmol), Na2CO3(100mg,0.92mmol) and a small amount of palladium chloride as a catalyst were mixed in 8mL of dioxane and stirred under N2The mixture was stirred at 100 ℃ for 12h with protection. Then spin-drying, and purifying by silica gel column chromatography to obtain a red solid product NY 3.
Example 3
The preparation method of the near-infrared viscosity probe NY3 based on the BD fluorophore comprises the following steps:
3.1 Compound 1: in a 100mL three-necked flask, 6-bromo-N, N-dimethylnaphthalen-2-amine (200mg, 0.80mmol), 4,4,4'4', 5,5,5',5' -octamethyl-2, 2' -bis (1,3, 2-dioxaborolane) (150mg, 0.60mmol) and CH3COOK (300mg, 3.0mmol) was added to a 15mL dioxane solution. After addition of the catalyst palladium chloride, the mixture is taken up in N2Stirring at 80 ℃ for 12h under protection. Then, the intermediate product, compound 1, was obtained as a white solid by extraction with EtOAc, spin-drying, and purification by silica gel column chromatography.
3.2 Compound 2: acetic acid (15mL) was heated to 70 ℃ and then NBD-Cl (0.66g,3mmol) and Fe powder (0.81g,0.45mmol) were added. In N2The mixture was stirred at 70 ℃ for 30min with protection. The reaction was cooled to room temperature and concentrated under reduced pressure. Dissolving the residue inIn ethyl acetate, NaHCO is used3Washing was carried out 3 times. The organic layers were combined and washed with anhydrous Na2SO4And (5) drying. The residue was purified by silica column (ethyl acetate/petroleum ether ═ 1/10) to give compound 2 as a yellow powder.
3.3 Compound 3: to a solution of compound 2(0.15g,3mmol) in acetonitrile (15mL) was added CH in sequence3I (0.036mL,0.5mmol) and Cs2CO3(900mg,3 mmol). The reaction was then stirred at 85 ℃ for 12 hours. The product was extracted with EtOAc (3X 75 mL). The organic layers were combined and the solvent was evaporated in vacuo. The residue was purified by silica column (petroleum ether/ethyl acetate ═ 20/1) to give compound 3 as an orange-red powder.
3.4 NY 3: mixing compound 1(300mg,1.08mmol), compound 3(150mg,0.6mmol), Na2CO3(300mg,2.76mmol) and a small amount of palladium chloride as a catalyst were mixed in 15mL of dioxane and stirred under N2The mixture was stirred at 100 ℃ for 12h with protection. Then spin-drying, and purifying by silica gel column chromatography to obtain a red solid product NY 3.
The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.
Claims (8)
1. The near-infrared fluorescent probe for viscosity detection is characterized by being a probe NY3 based on a BD (nitrobenzo-2-oxa-1, 3-diazole) fluorophore, and the structural formula of the fluorescent probe is shown as (I):
2. the preparation of the near-infrared fluorescent probe for detecting viscosity according to claim 1, wherein: the preparation method of the fluorescent probe NY3 comprises the following steps:
50-200mg of compound 1, 25-100m of compound 3g, 32.5-130mg of Na2CO3Mixed with a small amount of catalyst palladium chloride in 2-10mL dioxane and stirred in N2Stirring the mixture at 100 ℃ for 12h under protection, then spin-drying, and purifying by silica gel column chromatography to obtain a red solid product NY 3;
3. the method for preparing the near-infrared fluorescent probe for detecting viscosity according to claim 2, wherein the molar ratio of the compound 1 to the compound 3 in the preparation method of the fluorescent probe NY3 is 1.2:1, the compound 3 is not excessive during the reaction, otherwise byproducts are generated, the yield is reduced, the nitrogen protection is kept for the secondary reaction, and the reaction is in a high temperature environment, otherwise, other impurities are generated.
4. The method for preparing the near-infrared fluorescent probe for detecting viscosity according to claim 2, wherein the method for preparing the near-infrared probe precursor compound 1 for detecting viscosity comprises the following steps:
compound 1: in a 100mL three-necked flask, 6-bromo-N, N-dimethylnaphthalen-2-amine 126.0-504.0mg, 4,4,4',4',5,5,5',5' -octamethyl-2, 2' -bis (1,3, 2-dioxaborolane) 152.35-609.4mg and CH3COOK 93.15-372.6mg was added to 2-10mL of a dioxane solution, palladium chloride as a catalyst was added, and the mixture was stirred in N2Stirring at 80 deg.C for 12h under protection, extracting with EtOAc solution, spin drying, and purifying with silica gel column chromatography to obtain intermediate white solid compound 1, which has the following reaction formula:
5. the preparation of the near infrared probe for detecting viscosity according to claim 4, wherein: preparation of intermediate Compound 1 in preparation of two-photon Naphthol derivative Compound 6-bromo-N, N-dimethylNaphthalen-2-amine, 4,4,4',4',5,5,5',5' -octamethyl-2, 2' -bis (1,3, 2-dioxaborolane), CH3The molar ratio of COOK is 1: 1.2-2: 2-3, and the time is 12 hours.
6. The method for preparing the near-infrared probe for detecting viscosity according to claim 2, wherein the method for preparing the near-infrared probe precursor compound 3 for detecting viscosity comprises the following steps:
(1) compound 2: heating acetic acid 4.5-18mL to 70 deg.C, adding NBD-Cl 0.11-0.44g and Fe powder 0.09-0.36g, adding into the mixture under stirring2The mixture was stirred at 70 ℃ for 30min under protection, the reaction was cooled to room temperature and concentrated under reduced pressure, the residue was dissolved in ethyl acetate and washed with NaHCO3Washing 3 times, combining organic layers and adding anhydrous Na2SO4The residue was dried and purified by silica column (ethyl acetate/petroleum ether ═ 1/10) to give compound 2 as a yellow powder, according to the following reaction scheme:
(2) compound 3: to a solution of 0.025-0.1g of Compound 2 in L acetonitrile 2.5-10m in sequence CH3I0.036-0.144 mL and Cs2CO3150mg, the reaction was then stirred at 85 ℃ for 12h, the product was extracted with EtOAc 3X 75mL, the organic layers were combined, the solvent was evaporated in vacuo and the residue was purified by silica column (petroleum ether/ethyl acetate: 20/1) to give compound 3 as an orange-red powder according to the following reaction formula:
7. the preparation of the near infrared probe for detecting viscosity according to claim 6, wherein:
in the step (1), the mol ratio of NBD-Cl powder to Fe powder is 1:3-1:5, acetic acid is heated to 70 ℃ in advance, and then the mixture is stirred for 30min under the protection of nitrogen, otherwise, the yield is reduced;
compound 2, CH in step (2)3I、Cs2CO3All molar ratios of (1): 4-5: 3-5, in the reaction process, CH3The molar amount of I should not be too small, otherwise excessive by-products are produced, the yield is reduced, and secondly, the reaction must be protected by nitrogen gas, otherwise the reaction yield is reduced.
8. The use of the near-infrared probe for detecting viscosity according to claim 1, wherein the fluorescent probe is a probe NY3 based on a BD (nitrobenzo-2-oxa-1, 3-diazole) fluorophore, and the probe can be used for preparing a kit for detecting the change of intracellular viscosity in a physiological system.
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