CN116768837A - Benzopyran-2-one-based fluorescence chemical sensor and preparation method and application thereof - Google Patents
Benzopyran-2-one-based fluorescence chemical sensor and preparation method and application thereof Download PDFInfo
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- 239000000126 substance Substances 0.000 title claims abstract description 49
- FXRDPPFLWGSMQT-UHFFFAOYSA-N benzo[f]chromen-3-one Chemical compound C1=CC=C2C(C=CC(O3)=O)=C3C=CC2=C1 FXRDPPFLWGSMQT-UHFFFAOYSA-N 0.000 title claims abstract description 16
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- 102000003425 Tyrosinase Human genes 0.000 claims abstract description 37
- 108060008724 Tyrosinase Proteins 0.000 claims abstract description 37
- 239000003960 organic solvent Substances 0.000 claims abstract description 11
- 230000000694 effects Effects 0.000 claims abstract description 8
- 239000012043 crude product Substances 0.000 claims abstract description 7
- JHQONOCFAYZOEQ-UHFFFAOYSA-N 3-(bromomethyl)phenol Chemical compound OC1=CC=CC(CBr)=C1 JHQONOCFAYZOEQ-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000004440 column chromatography Methods 0.000 claims abstract description 4
- 239000003513 alkali Substances 0.000 claims abstract description 3
- 238000010438 heat treatment Methods 0.000 claims abstract 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 39
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 36
- 238000006243 chemical reaction Methods 0.000 claims description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical group [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 10
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 8
- 150000001875 compounds Chemical class 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 7
- BRQZHMHHZLRXOO-UHFFFAOYSA-N 3-acetyl-7-hydroxychromen-2-one Chemical compound C1=C(O)C=C2OC(=O)C(C(=O)C)=CC2=C1 BRQZHMHHZLRXOO-UHFFFAOYSA-N 0.000 claims description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 5
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 239000012046 mixed solvent Substances 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 239000008055 phosphate buffer solution Substances 0.000 claims description 5
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 4
- 239000003208 petroleum Substances 0.000 claims description 4
- 239000002585 base Substances 0.000 claims description 3
- FXHOOIRPVKKKFG-UHFFFAOYSA-N N,N-Dimethylacetamide Chemical compound CN(C)C(C)=O FXHOOIRPVKKKFG-UHFFFAOYSA-N 0.000 claims description 2
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 claims description 2
- 240000003768 Solanum lycopersicum Species 0.000 claims description 2
- 235000003953 Solanum lycopersicum var cerasiforme Nutrition 0.000 claims description 2
- 244000061456 Solanum tuberosum Species 0.000 claims description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 2
- 235000013405 beer Nutrition 0.000 claims description 2
- 235000020104 cherry wine Nutrition 0.000 claims description 2
- 235000012015 potatoes Nutrition 0.000 claims description 2
- 239000012312 sodium hydride Substances 0.000 claims description 2
- 229910000104 sodium hydride Inorganic materials 0.000 claims description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 2
- ZPSJGADGUYYRKE-UHFFFAOYSA-N 2H-pyran-2-one Chemical compound O=C1C=CC=CO1 ZPSJGADGUYYRKE-UHFFFAOYSA-N 0.000 claims 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims 1
- JIJWRJCVLPNOHM-UHFFFAOYSA-N bromo(phenyl)methanol Chemical compound OC(Br)C1=CC=CC=C1 JIJWRJCVLPNOHM-UHFFFAOYSA-N 0.000 claims 1
- 238000004458 analytical method Methods 0.000 abstract description 2
- 239000012472 biological sample Substances 0.000 abstract description 2
- 239000002904 solvent Substances 0.000 abstract description 2
- 239000012467 final product Substances 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
- 238000004809 thin layer chromatography Methods 0.000 description 6
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000002189 fluorescence spectrum Methods 0.000 description 4
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- IVJMJRRORVMRJJ-UHFFFAOYSA-N 7-hydroxy-4-phenylchromen-2-one Chemical compound C=1C(=O)OC2=CC(O)=CC=C2C=1C1=CC=CC=C1 IVJMJRRORVMRJJ-UHFFFAOYSA-N 0.000 description 1
- 206010001557 Albinism Diseases 0.000 description 1
- 102000030523 Catechol oxidase Human genes 0.000 description 1
- 108010031396 Catechol oxidase Proteins 0.000 description 1
- AHMIDUVKSGCHAU-UHFFFAOYSA-N Dopaquinone Natural products OC(=O)C(N)CC1=CC(=O)C(=O)C=C1 AHMIDUVKSGCHAU-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- WTDRDQBEARUVNC-LURJTMIESA-N L-DOPA Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-LURJTMIESA-N 0.000 description 1
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 description 1
- AHMIDUVKSGCHAU-LURJTMIESA-N L-dopaquinone Chemical compound [O-]C(=O)[C@@H]([NH3+])CC1=CC(=O)C(=O)C=C1 AHMIDUVKSGCHAU-LURJTMIESA-N 0.000 description 1
- 206010047642 Vitiligo Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229960004502 levodopa Drugs 0.000 description 1
- 230000008099 melanin synthesis Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
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- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
- C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
- C07D311/16—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted in position 7
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Abstract
Description
技术领域Technical field
本发明属于化学分析测试领域，具体涉及一种基于苯并吡喃-2-酮的荧光化学传感器及其制备方法和应用。The invention belongs to the field of chemical analysis and testing, and specifically relates to a fluorescent chemical sensor based on benzopyran-2-one and its preparation method and application.
背景技术Background technique
酪氨酸酶(TYR)是一种多酚氧化酶，广泛分布于动物、植物和微生物系统中。它在黑色素的生物合成中发挥重要作用，可以催化左旋多巴形成多巴醌，经过一系列反应最终形成黑色素，被认为是黑色素瘤的生物标志物。一旦酪氨酸酶的活性降低或合成出现异常都会严重影响黑色素的代谢，从而导致白癫疯、白化病等疾病的发生。因此，开发一种有效的检测方法，快速检测酪氨酸酶的含量有重要的意义。Tyrosinase (TYR) is a polyphenol oxidase that is widely distributed in animal, plant and microbial systems. It plays an important role in the biosynthesis of melanin and can catalyze the formation of dopaquinone from levodopa, which ultimately forms melanin through a series of reactions and is considered a biomarker of melanoma. Once the activity of tyrosinase is reduced or the synthesis is abnormal, it will seriously affect the metabolism of melanin, leading to the occurrence of vitiligo, albinism and other diseases. Therefore, it is of great significance to develop an effective detection method to quickly detect the content of tyrosinase.
目前，基于检测酪氨酸酶活性的荧光化学传感器的开发是研究的热点。酪氨酸酶在生物医药，食品工业，环境领域都用重要的作用，迄今为止，现有的荧光化学传感器的应用比较单一，多数以生物医药行业为主，很少体现在食品工业的应用。Currently, the development of fluorescent chemical sensors based on detecting tyrosinase activity is a hot research topic. Tyrosinase plays an important role in biomedicine, food industry, and environmental fields. So far, the applications of existing fluorescent chemical sensors are relatively single. Most of them are mainly used in the biomedicine industry and are rarely used in the food industry.
发明内容Contents of the invention
本发明提供了一种基于苯并吡喃-2-酮的荧光化学传感器，该荧光化学传感器的结构式为：The invention provides a fluorescent chemical sensor based on benzopyran-2-one. The structural formula of the fluorescent chemical sensor is:
本发明还提供了一种基于苯并吡喃-2-酮的荧光化学传感器的制备方法：制备该荧光化学传感器的化学反应式为：The invention also provides a method for preparing a fluorescent chemical sensor based on benzopyran-2-one: the chemical reaction formula for preparing the fluorescent chemical sensor is:
具体操作步骤为：将3-乙酰基-7羟基-苯并吡喃-2-酮和碱按1:3摩尔当量比加入到有机溶剂中搅拌10分钟，然后将2当量的间羟基苄基溴溶于有机溶剂中，并逐滴滴加到反应体系中，氮气保护条件下，0～60℃加热3～9小时，通过TLC监测反应结束后，得到粗品，通过乙酸乙酯和水萃取，收集乙酸乙酯层样品，加入无水硫酸钠干燥，减压除去有机溶剂，最后以体积比为2:1的石油醚和乙酸乙酯的混合溶剂作为展开剂，通过柱色谱法提纯，得到淡黄色的固体目标物化合物。The specific operation steps are: add 3-acetyl-7hydroxy-benzopyran-2-one and alkali to an organic solvent at a molar equivalent ratio of 1:3 and stir for 10 minutes, and then add 2 equivalents of m-hydroxybenzyl bromide Dissolve in an organic solvent, and add it dropwise to the reaction system. Under nitrogen protection, heat at 0 to 60°C for 3 to 9 hours. After the reaction is completed, monitor by TLC to obtain a crude product, which is extracted with ethyl acetate and water and collected. Take the ethyl acetate layer sample and dry it by adding anhydrous sodium sulfate. Remove the organic solvent under reduced pressure. Finally, use a mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 2:1 as the developing agent and purify it through column chromatography to obtain a light yellow color. solid target compounds.
碱为碳酸钾、氢化钠或N,N-二异丙基乙胺(DIEA)。The base is potassium carbonate, sodium hydride or N,N-diisopropylethylamine (DIEA).
有机溶剂为：N,N-二甲基甲酰胺、乙腈、N,N-二甲基乙酰胺、四氢呋喃。The organic solvents are: N,N-dimethylformamide, acetonitrile, N,N-dimethylacetamide, and tetrahydrofuran.
本发明还提供了一种上述荧光化学传感器的应用：基于苯并吡喃-2-酮的荧光化学传感器应用于酪氨酸酶的检测，直接在磷酸盐缓冲溶液(PBS缓冲液，浓度为10mM，pH＝7.4)中，通过荧光增强的方式检测酪氨酸酶，操作简便、选择性高。该荧光化学传感器本身几乎不显示荧光。当它在识别酪氨酸酶后，酪氨酸酶会使荧光化学传感器中的3-乙酰基-7-羟基-苯并吡喃-2-酮结构的荧光团释放，在455nm处的荧光强度有了显著的增强，从而达到了检测酪氨酸酶的目的，荧光强度的强弱与酪氨酸酶活性直接相关。The present invention also provides an application of the above-mentioned fluorescent chemical sensor: the fluorescent chemical sensor based on benzopyran-2-one is applied to the detection of tyrosinase, directly in phosphate buffer solution (PBS buffer solution, the concentration is 10mM , pH=7.4), tyrosinase is detected through fluorescence enhancement, which is easy to operate and highly selective. The fluorescent chemical sensor itself displays almost no fluorescence. When it recognizes tyrosinase, tyrosinase will release the fluorophore of the 3-acetyl-7-hydroxy-benzopyran-2-one structure in the fluorescent chemical sensor, and the fluorescence intensity at 455nm With significant enhancement, the purpose of detecting tyrosinase is achieved. The intensity of fluorescence is directly related to tyrosinase activity.
本发明荧光化学传感器用于啤酒、樱桃酒、西红柿、洋芋中的酪氨酸酶活性定量检测。The fluorescent chemical sensor of the present invention is used for quantitative detection of tyrosinase activity in beer, cherry wine, tomatoes, and potatoes.
本发明解决了荧光化学传感器在磷酸盐缓冲溶液体系中，对加入的酪氨酸酶的荧光识别问题。The invention solves the problem of fluorescent recognition of added tyrosinase by a fluorescent chemical sensor in a phosphate buffer solution system.
本发明的有益效果在于：本荧光化学传感器荧光背景低，对酪氨酸酶的响应效果好，能够在良溶剂中检测出酪氨酸酶，可以为在生物样本中的应用提供良好的前提条件。The beneficial effects of the present invention are: the fluorescent chemical sensor has low fluorescence background, good response to tyrosinase, can detect tyrosinase in a good solvent, and can provide good prerequisites for application in biological samples. .
附图说明：Picture description:
图1为实施例1制备的荧光化学传感器在PBS缓冲液中与酪氨酸酶作用前后的荧光光谱图。Figure 1 is a fluorescence spectrum chart of the fluorescent chemical sensor prepared in Example 1 before and after the interaction with tyrosinase in PBS buffer.
图2为实施例1制备的荧光化学传感器在PBS缓冲液中与不同浓度的酪氨酸酶作用后在455nm处的荧光光谱图。Figure 2 is a fluorescence spectrum at 455 nm of the fluorescent chemical sensor prepared in Example 1 after reacting with different concentrations of tyrosinase in PBS buffer.
图3为实施例1制备的荧光化学传感器在PBS缓冲液中在455nm处的动力学图。Figure 3 is a kinetic diagram of the fluorescent chemical sensor prepared in Example 1 in PBS buffer at 455 nm.
图4为实施例1制备的荧光化学传感器在PBS缓冲液中在455nm处对酪氨酸酶活性的拟合直线图。Figure 4 is a fitting straight line graph of the tyrosinase activity at 455 nm of the fluorescent chemical sensor prepared in Example 1 in PBS buffer.
图5为实施例1制备的荧光化学传感器的核磁氢谱图。Figure 5 is a hydrogen nuclear magnetic spectrum of the fluorescent chemical sensor prepared in Example 1.
图6为实施例1荧光化学传感器和对比实施例1传感器对酪氨酸酶识别作用的对比图。Figure 6 is a comparison chart of the recognition effects of the fluorescent chemical sensor of Example 1 and the sensor of Comparative Example 1 on tyrosinase.
图7为对比实施例1荧光化学传感器的核磁氢谱图。Figure 7 is a hydrogen nuclear magnetic spectrum of the fluorescent chemical sensor of Comparative Example 1.
具体实施方式Detailed ways
本发明结合下列实施例作进一步详述：The present invention is further described in detail in conjunction with the following examples:
实施例1Example 1
将3-乙酰基-7-羟基-苯并吡喃-2-酮(0.102g，0.5mmol)和无水碳酸钾(0.207g，1.5mmol)溶解于3mL N,N-二甲基甲酰胺(DMF)溶液中搅拌10分钟，然后将间羟基苄基溴(0.186g，1mmol)溶于0.5mL N,N-二甲基甲酰胺(DMF)，并逐滴滴加到反应体系中，氮气保护条件下，60℃加热6小时，通过TLC监测反应结束后，得到粗品，通过乙酸乙酯和水萃取，收集乙酸乙酯层样品，加入无水硫酸钠干燥，减压除去乙酸乙酯，最后以体积比为2:1的石油醚和乙酸乙酯的混合溶剂作为展开剂，通过柱色谱法提纯，得到淡黄色固体目标物化合物(荧光传感器1)0.0305g，收率19.7％。Dissolve 3-acetyl-7-hydroxy-benzopyran-2-one (0.102g, 0.5mmol) and anhydrous potassium carbonate (0.207g, 1.5mmol) in 3mL N,N-dimethylformamide ( DMF) solution for 10 minutes, then dissolve m-hydroxybenzyl bromide (0.186g, 1mmol) in 0.5mL N,N-dimethylformamide (DMF), and add it dropwise to the reaction system under nitrogen protection Under the conditions, heat at 60°C for 6 hours. After the reaction is monitored by TLC, the crude product is obtained. Extract with ethyl acetate and water. Collect the ethyl acetate layer sample, add anhydrous sodium sulfate to dry, remove the ethyl acetate under reduced pressure, and finally use A mixed solvent of petroleum ether and ethyl acetate with a volume ratio of 2:1 was used as a developing agent and purified by column chromatography to obtain 0.0305g of the target compound (fluorescence sensor 1) as a light yellow solid with a yield of 19.7%.
该荧光化学传感器在检测酪氨酸酶中的具体应用方法为：向96孔板中分别加入8μL浓度为10000U/mL的酪氨酸酶溶液、190μL的磷酸盐缓冲溶液和2μL浓度为3mM的荧光化学传感器，同时以不加酪氨酸酶(Blank)的含有3-乙酰基-7羟基-苯并吡喃-2-酮荧光化学传感器的溶液作为对比。将每孔充分混合均匀，使得每个孔中荧光化学传感器的浓度为0.03mM，酪氨酸酶的浓度为400U/mL，通过酶标仪检测出每孔中溶液的荧光强度。结果显示：荧光化学传感器本身几乎无荧光，当与酪氨酸酶反应后，荧光强度增强，在455nm处的荧光强度增加约20倍，说明该传感器是一种识别酪氨酸酶的荧光增强型化学传感器。The specific application method of this fluorescent chemical sensor in detecting tyrosinase is: add 8 μL of tyrosinase solution with a concentration of 10000U/mL, 190 μL of phosphate buffer solution and 2 μL of fluorescence with a concentration of 3mM into a 96-well plate. chemical sensor, and a solution containing a 3-acetyl-7hydroxy-benzopyran-2-one fluorescent chemical sensor without adding tyrosinase (Blank) was used as a comparison. Mix each well thoroughly so that the concentration of the fluorescent chemical sensor in each well is 0.03mM and the concentration of tyrosinase is 400U/mL. Use a microplate reader to detect the fluorescence intensity of the solution in each well. The results show that the fluorescent chemical sensor itself has almost no fluorescence. When reacting with tyrosinase, the fluorescence intensity is enhanced. The fluorescence intensity at 455nm increases by about 20 times, indicating that the sensor is a fluorescence-enhanced sensor that recognizes tyrosinase. chemical sensors.
图1为实施例1制备的荧光化学传感器在PBS缓冲溶液中与酪氨酸酶作用后的荧光光谱图。图中显示，荧光化学传感器本身几乎没有荧光，当与酪氨酸酶反应后，在455nm处的荧光强度发生了显著的升高。Figure 1 is a fluorescence spectrum diagram of the fluorescent chemical sensor prepared in Example 1 after reacting with tyrosinase in PBS buffer solution. The figure shows that the fluorescent chemical sensor itself has almost no fluorescence. When reacted with tyrosinase, the fluorescence intensity at 455nm increases significantly.
图2为实施例1制备的荧光化学传感器在PBS缓冲溶液中与不同浓度的酪氨酸酶作用后的荧光光谱图。图中显示，酪氨酸酶的浓度范围为20～90U/mL,随着酪氨酸酶浓度的降低，荧光化学传感器的荧光强度逐渐减弱。Figure 2 is a fluorescence spectrum chart of the fluorescent chemical sensor prepared in Example 1 after reacting with different concentrations of tyrosinase in PBS buffer solution. The figure shows that the concentration range of tyrosinase is 20-90U/mL. As the concentration of tyrosinase decreases, the fluorescence intensity of the fluorescent chemical sensor gradually weakens.
图3为实施例1制备的基于苯并吡喃-2-酮荧光化学传感器的核磁氢谱。1HNMR(400MHz,DMSO-d6)δ9.51(s,1H),8.64(s,1H),7.89(d,J＝8.7Hz,1H),7.19(t,J＝7.8Hz,1H),7.13(d,J＝2.2Hz,1H),7.08(dd,J＝8.7,2.3Hz,1H),6.90–6.84(m,2H),6.73(dd,J＝8.0,2.4Hz,1H),5.19(s,2H),2.56(s,3H).Figure 3 is the hydrogen nuclear magnetic spectrum of the benzopyran-2-one based fluorescent chemical sensor prepared in Example 1. 1 HNMR (400MHz, DMSO-d 6 ) δ9.51 (s, 1H), 8.64 (s, 1H), 7.89 (d, J = 8.7Hz, 1H), 7.19 (t, J = 7.8Hz, 1H), 7.13(d,J＝2.2Hz,1H),7.08(dd,J＝8.7,2.3Hz,1H),6.90–6.84(m,2H),6.73(dd,J＝8.0,2.4Hz,1H),5.19 (s,2H),2.56(s,3H).
实施例2Example 2
将3-乙酰基-7羟基-苯并吡喃-2-酮(0.143g，0.7mmol)和无水碳酸钾(0.289g，2.1mmol)溶解于4mL N,N-二甲基甲酰胺(DMF)溶液中搅拌10分钟，然后将间羟基苄基溴(0.261g，1.4mmol)溶于1mL N,N-二甲基甲酰胺(DMF)，并逐滴滴加到反应体系中，氮气保护条件下，60℃加热9小时，通过TLC监测反应结束后，得到粗品，通过乙酸乙酯和水萃取，收集乙酸乙酯层样品，加入无水硫酸钠干燥，减压除去有机溶剂，最后以体积比为2:1的石油醚和乙酸乙酯的混合溶剂作为展开剂，通过薄层色谱法提纯，得到淡黄色固体目标物化合物0.0219g，收率10.1％。Dissolve 3-acetyl-7hydroxy-benzopyran-2-one (0.143g, 0.7mmol) and anhydrous potassium carbonate (0.289g, 2.1mmol) in 4mL N,N-dimethylformamide (DMF ) solution for 10 minutes, then dissolve m-hydroxybenzyl bromide (0.261g, 1.4mmol) in 1mL N,N-dimethylformamide (DMF), and add it dropwise to the reaction system under nitrogen protection conditions , and heated at 60°C for 9 hours. After the reaction was monitored by TLC, the crude product was obtained, and extracted with ethyl acetate and water. Collect the ethyl acetate layer sample, add anhydrous sodium sulfate to dry it, remove the organic solvent under reduced pressure, and finally use the volume ratio A 2:1 mixed solvent of petroleum ether and ethyl acetate was used as a developing agent and purified by thin layer chromatography to obtain 0.0219g of the target compound as a light yellow solid with a yield of 10.1%.
实施例3Example 3
为提高收率对反应条件进行了优化：The reaction conditions were optimized to improve the yield:
对比实施例1Comparative Example 1
将7-羟基-4-苯基香豆素(0.239g，1mmol)和无水碳酸钾(0.415g，3mmol)溶解于4mL N,N-二甲基甲酰胺(DMF)溶液中搅拌10分钟，然后将间羟基苄基溴(0.373g，2mmol)溶于1mL N,N-二甲基甲酰胺(DMF)，并逐滴滴加到反应体系中，氮气保护条件下，60℃加热6小时，通过TLC监测反应结束后，得到粗品，通过二氯甲烷和水萃取，收集二氯甲烷层样品，加入无水硫酸钠干燥，减压除去有机溶剂，最后以展开剂为体积比为100:1的二氯甲烷和甲醇的混合溶剂，通过薄层色谱法提纯，得到淡黄色固体目标物化合物(荧光传感器2)0.055g，收率16.1％，得到的化合物结构如下式：Dissolve 7-hydroxy-4-phenylcoumarin (0.239g, 1mmol) and anhydrous potassium carbonate (0.415g, 3mmol) in 4mL N,N-dimethylformamide (DMF) solution and stir for 10 minutes. Then, m-hydroxybenzyl bromide (0.373g, 2mmol) was dissolved in 1mL N,N-dimethylformamide (DMF), and added dropwise to the reaction system, and heated at 60°C for 6 hours under nitrogen protection. After the reaction was monitored by TLC, the crude product was obtained, extracted with dichloromethane and water, collected dichloromethane layer samples, added anhydrous sodium sulfate to dry, removed the organic solvent under reduced pressure, and finally used the developing agent as a volume ratio of 100:1 The mixed solvent of methylene chloride and methanol was purified by thin layer chromatography to obtain 0.055g of the target compound (fluorescence sensor 2) as a light yellow solid, with a yield of 16.1%. The structure of the obtained compound is as follows:
1H NMR(400MHz,DMSO-d6)δ9.53(s,1H),7.59–7.51(m,5H),7.35(d,J＝8.9Hz,1H),7.22–7.14(m,2H),7.01–6.97(m,1H),6.89–6.83(m,2H),6.72(d,J＝8.1Hz,1H),6.24(s,1H),5.16(s,2H). 1 H NMR (400MHz, DMSO-d 6 ) δ9.53 (s, 1H), 7.59–7.51 (m, 5H), 7.35 (d, J = 8.9Hz, 1H), 7.22–7.14 (m, 2H), 7.01–6.97(m,1H),6.89–6.83(m,2H),6.72(d,J＝8.1Hz,1H),6.24(s,1H),5.16(s,2H).
合成的荧光传感器2在实施例1相同条件下加入酪氨酸酶的前后荧光强度几乎不发生变化。The fluorescence intensity of the synthesized fluorescence sensor 2 almost did not change before and after adding tyrosinase under the same conditions as in Example 1.
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