CN116041353A - 一种超氧阴离子自由基化学发光探针及其制备方法和在阿尔茨海默症早期诊断中的应用 - Google Patents
一种超氧阴离子自由基化学发光探针及其制备方法和在阿尔茨海默症早期诊断中的应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及一种探针及其制备方法和应用,具体涉及一种超氧阴离子自由基化学发光探针及其制备方法和在阿尔茨海默症早期诊断中的应用。
背景技术
阿尔茨海默症(AD)是一种常见的神经退行性疾病,主要在老年人群中盛行,影响老年的生活质量和健康状况。大量数据表明大脑内的氧化应激与AD有密不可分的关系,而且相关研究显示AD大脑内活性氧(Reactive Oxygen Species,ROS)的水平要高于正常大脑。而ROS的产生与大脑内线粒体损伤、营养物质的氧化、Aβ与Tau蛋白的沉积以及炎症等均有关联,因此ROS被认为是AD的另一大致病因素。超氧阴离子自由基(O2 ·-)作为细胞内最先产生的一类ROS,是体内其他ROS的主要来源,在众多ROS中具有重要的作用和地位。因此监测O2 ·-的浓度变化对于揭示AD的发病机制具有至关重要的作用。
本发明将设计一类性质稳定,能够高灵敏及高信噪比监测O2 ·-浓度变化的化学发光探针。并且利用探针对O2 ·-的高灵敏检测实现对AD的早期检测以及评价药物疗效。
目前检测O2 ·-的方法主要包括电子顺磁共振法、高效液相色谱法、电化学法以及光学成像法等。
发明内容
发明目的:本发明的目的在于提供一种超氧阴离子自由基化学发光探针,该探针可用于检测O2 ·-,并且可以通过血脑屏障。本发明还有一个目的是提供所述超氧阴离子自由基化学发光探针的制备方法以及其在阿尔茨海默症早期诊断中的应用。
技术方案:本发明所述的用于超氧阴离子自由基检测的化学发光探针,其结构式如式(I)所示:
其中A选自饱和或不饱和的杂环基、芳基和杂芳基;
n=1,2,3,4;
R1为氢、烷基、氰烷基、取代的5-6元饱和或不饱和的芳基、杂环基;
式中X=O、S、Se。
所述的化学发光探针,所述A选自苯基。
所述的化学发光探针,所述n=1,2。
所述的化学发光探针,所述化学发光探针选自:
所述的化学发光探针的制备方法,包括以下步骤:
所述的化学发光探针的制备方法,包括以下步骤:
(1)将2-(2-甲基-4H-苯并吡喃-4-亚基)丙二腈和(5-甲酰基-吡嗪-2-基)-氨基甲酸叔丁酯溶于乙腈,并滴加哌啶,在惰性气体保护下搅拌反应,反应结束后,将反应液冷却至室温,形成的固体经过冷却过滤洗涤,得到中间体化合物1;
(2)将中间体化合物1溶于三氟乙酸和二氯甲烷中,在室温下搅拌,反应结束后,将反应液中的二氯甲烷除去,再用油泵除去剩余的三氟乙酸,得到中间体化合物2;
(3)将中间体化合物2溶于乙醇中,然后加入丙酮醛和盐酸,在惰性气体保护下搅拌反应,反应结束后冷却至室温,加入***形成的固体经过冷却过滤洗涤,得到探针DIP-O;
或,
(4)将2-(2-甲基-4H-1-苯并噻喃-4-亚基)丙二腈,和(5-甲酰基-吡嗪-2-基)-氨基甲酸叔丁酯溶于乙腈,并滴加哌啶,在惰性气体保护下搅拌反应,反应结束后,将反应液冷却至室温,形成的固体经过冷却过滤洗涤,得到中间体化合物3;
(5)将中间体化合物3溶于三氟乙酸和二氯甲烷中,在室温下搅拌,反应结束后,将反应液中的二氯甲烷除去,再用油泵除去剩余的三氟乙酸,得到中间体化合物4;
(6)将中间体化合物4溶于乙醇中,然后加入丙酮醛和盐酸,在惰性气体保护下搅拌反应,反应结束后冷却至室温,加入***形成的固体经过冷却过滤洗涤,得到探针DIP-S;
或,
(7)将2-(2-甲基-4H-1-苯硒啉-4-亚基)丙二腈,和(5-甲酰基-吡嗪-2-基)-氨基甲酸叔丁酯溶于乙腈,并滴加哌啶,在惰性气体保护下搅拌反应,反应结束后,将反应液冷却至室温,形成的固体经过冷却过滤洗涤,得到中间体化合物5;
(8)将中间体化合物5溶于三氟乙酸和二氯甲烷中,在室温下搅拌,反应结束后,将反应液中的二氯甲烷除去,再用油泵除去剩余的三氟乙酸,得到中间体化合物6;
(9)将中间体化合物6溶于乙醇中,然后加入丙酮醛和盐酸,在惰性气体保护下搅拌反应,反应结束后冷却至室温,加入***形成的固体经过冷却过滤洗涤,得到探针DIP-Se。
所述的化学发光探针在制备超氧阴离子自由基的高灵敏检测中的应用。
所述的化学发光探针在制备阿尔茨海默症诊断试剂中的应用。
有益效果:本发明与现有技术相比,具有如下优点:本发明所述的化学发光探针能够克服已报道探针波长短,不过脑,灵敏度低等缺点,为O2 ·-化学发光探针的制备提供思路。实现对AD发展及治疗过程中大脑内O2 ·-变化的实时监测,可以明确O2 ·-作为AD生物标记物的可能性,对AD的诊断和治疗具有重要意义。
附图说明
图1为DIP-O、DIP-S、DIP-Se探针对KO2的响应;
图2为DIP-O探针对不同活性氧的选择性;
图3为DIP-O探针与LPS诱导炎症小鼠体内成像图;(右:PBS对照;中:LPS炎症;左:LPS+Tiron);
图4为DIP-O探针与APP和WT小鼠体内成像图(右:WT;左:APP)。
具体实施方式
合成本发明化合物的反应路线如下:
实施例1化学发光探针的制备
1、中间体化合物1的合成:
将2-(2-甲基-4H-苯并吡喃-4-亚基)丙二腈(56mg,0.27mmol)和(5-甲酰基-吡嗪-2-基)-氨基甲酸叔丁酯(60mg,0.27mmol)溶于乙腈,并滴加2滴哌啶,除氧后用氮气保护,在摄氏度下搅拌、回流反应7小时,用石油醚:乙酸乙酯(3∶1)点板,反应结束后,将反应液冷却至室温,反应液中析出暗红色沉淀,过滤并用乙腈洗涤沉淀,得到化合物1。
2、中间体化合物2的合成:
将中间体1(40mg,0.056mmol)溶于2mL三氟乙酸和0.5ml二氯甲烷中,在室温下搅拌0.5小时,用石油醚∶乙酸乙酯(1∶1)点板,反应结束后,将反应液中的二氯甲烷除去,再用油泵除去剩余的三氟乙酸,得到化合物2。
3、化合物DIP-O的合成:
称取中间体化合物2(10mg)加入厚壁加压瓶中,加入600μL乙醇使其溶解,再加入100μL丙酮醛,然后加入2~3滴盐酸,除氧、避光,71摄氏度回流12小时,待反应结束后冷却至室温,向其加入适量***,反应液中析出固体,过滤并用***洗涤得到探针DIP-O。1H NMR(300MHz,DMSO-d6)δ10.25(s,1H),δ7.72(d,J=7.2Hz,1H),7.64(dd,J=7.8,1.5Hz,1H),7.58(s,1H),7.50(dd,J=8.1,1H),7.41-7.37(m,1H),7.30(td,J=7.5,1H),7.25(d,1H),6.68(s,1H),6.40(d,J=16.7Hz,1H),1.58(s,3H).
4、化合物3的合成:
将2-(2-甲基-4H-1-苯并噻喃-4-亚基)丙二腈(56mg,0.25mmol)和(5-甲酰基-吡嗪-2-基)-氨基甲酸叔丁酯(60mg,0.27mmol)溶于乙腈,并滴加2滴哌啶,除氧后用氮气保护,在70摄氏度下搅拌、回流反应7小时,用石油醚∶乙酸乙酯(3∶1)点板,反应结束后,将反应液冷却至室温,反应液中析出暗红色沉淀,过滤并用乙腈洗涤沉淀,得到化合物3。
5、化合物4的合成
将中间体1(40mg,0.054mmol)溶于2mL三氟乙酸和0.5mL二氯甲烷中,在室温下搅拌0.5小时,用石油醚∶乙酸乙酯(1∶1)点板,反应结束后,将反应液中的二氯甲烷除去,再用油泵除去剩余的三氟乙酸,得到化合物4。
6、化合物DIP-S的合成:
称取中间体化合物4(10mg)加入厚壁加压瓶中,加入600μL乙醇使其溶解,再加入100μL丙酮醛,然后加入2~3滴盐酸,除氧、避光,71摄氏度回流12小时,待反应结束后冷却至室温,向其加入适量***,反应液中析出固体,过滤并用***洗涤得到探针DIP-S。1H NMR(300MHz,DMSO-d6)δ11.86(s,1H),8.60(d,J=14.8Hz,1H),7.78(dd,J=7.5,1.3Hz,1H),7.75-7.69(m,2H),7.56(td,J=7.7,1.3Hz,1H),7.51-7.44(m,2H),7.14(s,1H),6.57(d,J=14.9Hz,1H),1.77(s,3H).
7、化合物5的合成
将2-(2-甲基-4H-1-苯硒啉-4-亚基)丙二腈(56mg,0.23mmol)和(5-甲酰基-吡嗪-2-基)-氨基甲酸叔丁酯(60mg,0.27mmol)溶于乙腈,并滴加2滴哌啶,除氧后用氮气保护,在70摄氏度下搅拌、回流反应7小时,用石油醚∶乙酸乙酯(3∶1)点板,反应结束后,将反应液冷却至室温,反应液中析出暗红色沉淀,过滤并用乙腈洗涤沉淀,得到化合物5。
8、化合物6的合成
将中间体1(40mg,0.054mmol)溶于2mL三氟乙酸和0.5mL二氯甲烷中,在室温下搅拌0.5小时,用石油醚∶乙酸乙酯(1∶1)点板,反应结束后,将反应液中的二氯甲烷除去,再用油泵除去剩余的三氟乙酸,得到化合物6。
9、化合物DIP-Se的合成
称取中间体化合物6(10mg)加入厚壁加压瓶中,加入600μL乙醇使其溶解,再加入100μL丙酮醛,然后加入2~3滴盐酸,除氧、避光,71摄氏度回流12小时,待反应结束后冷却至室温,向其加入适量***,反应液中析出固体,过滤并用***洗涤得到探针DIP-Se。1HNMR(300MHz,DMSO-d6)δ10.14(s,1H),8.72-8.44(m,1H),8.28-8.01(m,2H),7.81-7.52(m,3H),7.44(s,1H),7.27(s,1H),7.10(s,1H),1.20(s,3H).
实施例2
称取实施例1制备的探针DIP-O 1mg溶于1mL DMSO溶液中,配制成浓度为2.7mM的标准液,用PBS缓冲液稀释到终浓度为25μM。称取实施例制备的探针DIP-S 1mg溶于1mLDMSO溶液中,配制成浓度为2.6mM的标准液,用PBS缓冲液稀释到终浓度为25μM。称取实施例制备的探针DIP-Se 1mg溶于1mL DMSO溶液中,配制成浓度为2.4mM的标准液,用PBS缓冲液稀释到终浓度为25μM。称取7.1mg KO2溶于DNSO溶液中,配置成10mM标准液,然后稀释成1mM标准液,使用UV分光光度法测其吸光度,O2 ·-在DMSO中的消光系数为2686±29,经过计算得到1mM溶于DMSO中的KO2中含有0.5mM的O2 ·-,再将标准液稀释成25μM的O2 ·-,分别加入到实施例制备的三种探针中,得到不同的化学发光强度,如图1所示。
实施例3
称取实施例1制备的探针DIP-O 1mg溶于1mL DMSO溶液中,配制成浓度为2.7mM的标准液;用PBS缓冲液稀释到终浓度为250nm并放置于96孔板中。然后分别加入不同的ROS,包括羟基自由基、亚硝酸根离子、次氯酸根离子、硫化氢、单线态氧、过氧化氢、超氧阴离子自由基。经验证,如图2所示,实施例制备的探针DIP-O与其它的分析物基本没有引起检测体系化学发光强度的变化。说明探针对超氧阴离子自由基具有非常高的选择性,可用于近一步生物成像。
实施例4
称取实施例1制备的探针DIP-O 1mg溶于15%DMSO、15%蓖麻油、70%PBS缓冲液。将2.5mg/mL LPS注射到一只小鼠腹腔建造小鼠腹腔模型,4h后腹腔注射提前配制好的探针药物;将2.5mg/mL LPS注射到一只小鼠腹腔建造小鼠腹腔模型,3h后腹腔注射抑制剂Tiron50μM/mL,1h后腹腔注射探针药物;对照组注射PBS缓冲溶液,4h同样注射探针。通过小动物活体光学成像***,可以看到对照小鼠腹部仅有可忽略不计的化学发光信号,炎症小鼠腹部具有明显的化学发光信号,而注射了抑制剂的小鼠化学发光信号较炎症小鼠信号减弱,如图3所示。说明探针对炎症产生的超氧阴离子自由基具有较高的灵敏性,可以快速与之发生化学反应从而产生化学发光。
实施例5
称取实施例1制备的探针DIP-O 1mg溶于15%DMSO、15%蓖麻油、70%PBS缓冲液中作为药物注射给阿尔茨海默症小鼠以及野生型小鼠,注射剂量为25mg/μL。APP小鼠和WT小鼠经麻醉后通过尾静脉分别注射相对应的剂量,通过小动物活体光学成像***,可以看到探针通过尾静脉注射到小鼠体内可以迅速到达小鼠脑袋并产生化学发光,当8min时化学发光达到最大强度,且APP组与WT组信号差异达到最大。如图4所示。说明探针可迅速到达指定部位,并与超氧阴离子自由基结合并产生化学发光。
Claims (8)
2.如权利要求1所述的化学发光探针,其特征在于,所述A选自苯基。
3.如权利要求1所述的化学发光探针,其特征在于,所述n=1,2。
6.如权利要求4所述的化学发光探针的制备方法,其特征在于,包括以下步骤:
(1)将2-(2-甲基-4H-苯并吡喃-4-亚基)丙二腈和(5-甲酰基-吡嗪-2-基)-氨基甲酸叔丁酯溶于乙腈,并滴加哌啶,在惰性气体保护下搅拌反应,反应结束后,将反应液冷却至室温,形成的固体经过冷却过滤洗涤,得到中间体化合物1;
(2)将中间体化合物1溶于三氟乙酸和二氯甲烷中,在室温下搅拌,反应结束后,将反应液中的二氯甲烷除去,再用油泵除去剩余的三氟乙酸,得到中间体化合物2;
(3)将中间体化合物2溶于乙醇中,然后加入丙酮醛和盐酸,在惰性气体保护下搅拌反应,反应结束后冷却至室温,加入***形成的固体经过冷却过滤洗涤,得到探针DIP-O;
或,
(4)将2-(2-甲基-4H-1-苯并噻喃-4-亚基)丙二腈,和(5-甲酰基-吡嗪-2-基)-氨基甲酸叔丁酯溶于乙腈,并滴加哌啶,在惰性气体保护下搅拌反应,反应结束后,将反应液冷却至室温,形成的固体经过冷却过滤洗涤,得到中间体化合物3;
(5)将中间体化合物3溶于三氟乙酸和二氯甲烷中,在室温下搅拌,反应结束后,将反应液中的二氯甲烷除去,再用油泵除去剩余的三氟乙酸,得到中间体化合物4;
(6)将中间体化合物4溶于乙醇中,然后加入丙酮醛和盐酸,在惰性气体保护下搅拌反应,反应结束后冷却至室温,加入***形成的固体经过冷却过滤洗涤,得到探针DIP-S;
或,
(7)2-(2-甲基-4H-1-苯硒啉-4-亚基)丙二腈,和(5-甲酰基-吡嗪-2-基)-氨基甲酸叔丁酯溶于乙腈,并滴加哌啶,在惰性气体保护下搅拌反应,反应结束后,将反应液冷却至室温,形成的固体经过冷却过滤洗涤,得到中间体化合物5;
(8)将中间体化合物5溶于三氟乙酸和二氯甲烷中,在室温下搅拌,反应结束后,将反应液中的二氯甲烷除去,再用油泵除去剩余的三氟乙酸,得到中间体化合物6;
(9)将中间体化合物6溶于乙醇中,然后加入丙酮醛和盐酸,在惰性气体保护下搅拌反应,反应结束后冷却至室温,加入***形成的固体经过冷却过滤洗涤,得到探针DIP-Se。
7.权利要求1所述的化学发光探针在制备超氧阴离子自由基的高灵敏检测中的应用。
8.权利要求1所述的化学发光探针在制备阿尔茨海默症诊断试剂中的应用。
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