CN109369668B - 一种荧光素衍生物dcct及其制备方法和应用 - Google Patents
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Abstract
本发明提供了一种荧光素衍生物DCCT及其制备方法和应用,所述荧光素衍生物,中文名称为O,O'‑(2',7'‑二氯荧光素‑3',6'‑二基)S,S'‑二‑对‑甲苯基双(碳酸酯),英文名称为O,O'‑(2',7'‑dichlorofluorescein‑3',6'‑diyl)S,S'‑di‑p‑tolyl bis(carbonothioate),缩写为DCCT。本发明提供了一种对去甲肾上腺素特异性检测的方法,是基于荧光素衍生物DCCT,在PB/DMSO(v/v,1:1,pH5.0)溶液中检测去甲肾上腺素。本发明DCCT可通过荧光成像技术检测脑组织切片中的去甲肾上腺素,在生物分子检测领域具有广阔的应用前景。
Description
技术领域
本发明涉及杂环化合物荧光探针,具体属于一种荧光素衍生物DCCT及其制备方法,以及该衍生物DCCT在脑组织切片去甲肾上腺素检测中的应用。
背景技术
去甲肾上腺素是交感神经系统中最重要的神经递质之一,在所有前脑活动如感知、记忆、学习和体内平衡中起着关键作用。去甲肾上腺素和与其结构类似的多巴胺和肾上腺素共存于交感神经系统。在神经元中,三种儿茶酚胺通过连续的酶促反应由酪氨酸合成,并以高浓度储存在酸性突触小泡(pH 5.0-5.5)中。
去甲肾上腺素功能障碍与神经退行性疾病和中枢神经系统疾病密切相关。例如,黑质中去甲肾上腺素能神经元的退化通常先于多巴胺能神经元的丢失。此外,去甲肾上腺素在临床中已被用作一线血管加压剂治疗休克。近年来,社会压力的增大等因素使得抑郁症患者数量激增。研究表明,五分之一的人在其一生中会经历重度抑郁症。目前,普遍认为重度抑郁症的内因是脑组织内去甲肾上腺素水平降低。对去甲肾上腺素的生理学和病理学作用研究受到越来越多的关注。发展荧光探针对脑组织中去甲肾上腺素分布的标记检测对其生理学和病理学作用研究具有重要的意义。
本发明提供了一种荧光素衍生物DCCT及其制备方法,并基于该衍生物DCCT实现了对脑组织中去甲肾上腺素的特异性检测。
发明内容
本发明的目的在于提供一种荧光素衍生物及其制备方法,以及该衍生物可作为检测试剂用于脑组织去甲肾上腺素的荧光检测;在检测去甲肾上腺素时选择性高,灵敏度高。
本发明提供的一种荧光素衍生物,中文名称为O,O'-(2',7'-二氯荧光素-3',6'-二基)S,S'-二-对-甲苯基双(碳酸酯),英文名称为O,O'-(2',7'-dichlorofluorescein-3',6'-diyl)S,S'-di-p-tolylbis(carbonothioate),缩写为DCCT,结构式为:
DCCT在PB/DMSO(v/v,1:1,pH 5.0)溶液中对去甲肾上腺素显示了优良的灵敏性和选择性,且检测过程简便。
DCCT的制备方法,包括如下步骤:
1)将4-甲基苯硫酚、三光气按摩尔比为2:1溶于二氯甲烷中,冰浴下逐滴加入与4-甲基苯硫酚等摩尔量的吡啶,于0℃反应1h后,混合物倒入水中,分液得有机相;有机相经水洗涤、干燥、浓缩得到S-(对甲苯基)碳酰氯,直接用于下一步合成;
2)将2',7'-二氯荧光素、三乙胺按摩尔比为1:2溶于二氯甲烷中,冰浴下将步骤1)所得与三乙胺等摩尔量的S-(对甲苯基)碳酰氯的二氯甲烷溶液加入体系中,体系逐渐升温至室温并继续反应10h;体系减压蒸干后柱色谱分离(洗脱剂配比为乙酸乙酯:石油醚=1:6)得到目标化合物DCCT。
本发明荧光素衍生物DCCT的用途:该衍生物可以在pH 5.0体系中对水环境和组织切片中的去甲肾上腺素特异性检测;所述的检测包含荧光检测和组织成像检测。
与现有技术相比,本发明具有如下有益效果:
1)所述荧光素衍生物合成简单,成本低廉,易于规模生产;
2)所述检测方法能够利用去甲肾上腺素与荧光探针的连续亲核取代反应并释放荧光信号实现去甲肾上腺素的特异性检测,其它神经递质及氨基酸不干扰对去甲肾上腺素的测定;
3)所述检测方法与传统免疫荧光标记法在对组织中去甲肾上腺素标记具有同等的特异性且操作简易、成本低廉。
附图说明:
图1 DCCT氢谱。
图2 DCCT碳谱。
图3 DCCT质谱。
图4实施例2DCCT与去甲肾上腺素作用的随时间荧光发射图。
图5实施例3DCCT与各种分析物作用的荧光发射图。其中1.空白;2.Gly;3.Glu;4.GSH;5.Lys;6.The;7.Cys;8.多巴胺;9.肾上腺素;10.去甲肾上腺素。
图6实施例4DCCT对小鼠脑组织中去甲肾上腺素进行特异性成像图。其中,1.商业DBH免疫荧光标记染料标记的去甲肾上腺素能细胞;2.DCCT染色成像;3.商业PNMT免疫荧光标记染料标记的肾上腺素能细胞;4.1和2相关性;5.明场成像。
具体实施方式:
下面结合实施例和附图对本发明做进一步说明,但本发明不受下述实施例的限制。
实施例1
DCCT的制备及表征:
将4-甲基苯硫酚(10mmol,1.24g)、三光气(5mmol,1.48g)溶于二氯甲烷中,冰浴下逐滴加入吡啶(10mmol,0.79g),于0℃反应1h后,混合物倒入水中,分液得有机相;有机相经水洗涤、干燥、浓缩得到S-(对甲苯基)碳酰氯,直接用于下一步合成;
将2',7'-二氯荧光素(1mmol,0.4g)、三乙胺(2mmol,0.2g)溶于二氯甲烷中,冰浴下将步骤1)所得的S-(对甲苯基)碳酰氯(2mmol,0.36g)的二氯甲烷溶液加入体系中,体系逐渐升温至室温并继续反应10h;体系减压蒸干后柱色谱分离(洗脱剂配比为乙酸乙酯:石油醚=1:6)得到目标化合物DCCT。
1H NMR(600MHz,DMSO-d6)δ8.05(d,J=7.6Hz,1H),7.86–7.74(m,4H),7.54(d,J=8.1Hz,4H),7.46(d,J=7.6Hz,1H),7.32(d,J=8.1Hz,4H),7.12(s,2H),2.35(s,6H)(图1)。13CNMR(151MHz,DMSO-d6)δ168.4,167.9,151.7,149.7,148.6,141.0,136.6,135.6,135.2,131.4,130.8,129.4,126.1,125.8,124.5,122.6,122.1,118.9,113.8,79.9,21.3(图2)。HRMS[M+H]+:m/z Calcd 701.0257,Found 701.0259(图3)。
实施例2
配制pH=5.0的PB缓冲溶液,配制2mM DCCT的DMSO溶液,配制100mM去甲肾上腺素的水溶液(含有50mM Na2S2O3和120mM NaCl);取2mL的PB/DMSO(v/v,1:1,pH 5.0)溶液、5μLDCCT的DMSO溶液加到一个荧光比色皿中,加入100μL去甲肾上腺素的水溶液,随时间在荧光分光光度仪上检测(510nm激发)。0-320min内,530nm的荧光强度逐渐增强,荧光发射图见图4。
实施例3
配制pH=5.0的PB缓冲溶液,配制2mM DCCT的DMSO溶液,分别配制100mM去甲肾上腺素、肾上腺素、多巴胺的水溶液(含有50mM Na2S2O3和120mM NaCl)、100mM Gly、Glu、GSH的水溶液、10mM Lys、The的水溶液和1mM Cys的水溶液;在10个荧光比色皿中,各加入2mL的PB/DMSO(v/v,1:1,pH 5.0)溶液、5μL DCCT的DMSO溶液,再分别加入100μL的水、Gly、Glu、GSH、Lys、The、Cys、多巴胺、肾上腺素和去甲肾上腺素。320min后在荧光分光光度仪上检测(510nm激发),(见图5)。去甲肾上腺素使得检测体系在530nm处荧光强度明显升高,其它的分析物没有引起检测体系荧光强度的变化。
经实验证明,其它分析物不干扰体系对去甲肾上腺素的检测。
实施例4
配制2mM DCCT的DMSO溶液,取小鼠冠状组织切片(40μm),于37℃依次孵育RabbitAnti-PNMT Polyclonal Antibody(1:500,bs-3912R;Bioss Antibodies)和Anti-DopamineβHydroxylase(1:1000,MAB308;Millipore)的混合物2h、Goat Anti-Rabbit IgG/Cy3(1:250,bs-0295G-Cy3;Bioss Antibodies)和Goat Anti-Mouse IgG/ALexa Fluor 350(1:100,bs-0296G-AF350;Bioss Antibodies)的混合物30min、DCCT(5μM,30min)后用激光共聚焦显微镜成像(见图6)。分别用405nm、500nm和548nm作为蓝色通道(430-480nm)、绿色通道(510-560nm)和红色通道(560-610nm)的激发光。所得成像蓝色通道和红色通道信号高度重叠,皮尔森系数为0.84。因此,DCCT可以对脑组织中去甲肾上腺素特异性标记。
Claims (6)
2.如权利要求1所述的一种荧光素衍生物DCCT的制备方法,其特征在于,包括如下步骤:
1)将4-甲基苯硫酚、三光气溶于二氯甲烷中,冰浴下逐滴加入吡啶,于0℃反应1h后,混合物倒入水中,分液得有机相;有机相经水洗涤、干燥、浓缩得到S-(对甲苯基)碳酰氯,直接用于下一步合成;
2)取2',7'-二氯荧光素、三乙胺溶于二氯甲烷中,冰浴下将步骤1)所得S-(对甲苯基)碳酰氯的二氯甲烷溶液加入体系中,体系逐渐升温至室温并继续反应10h;体系减压蒸干后柱色谱分离得到目标化合物DCCT。
3.如权利要求2所述的荧光素衍生物DCCT的制备方法,其特征在于,所述步骤1)中4-甲基苯硫酚、三光气和吡啶的摩尔比为2:1:2。
4.如权利要求2所述的荧光素衍生物DCCT的制备方法,其特征在于,所述步骤2)中S-(对甲苯基)碳酰氯、2',7'-二氯荧光素和三乙胺的摩尔比为2:1:2。
5.如权利要求2所述的荧光素衍生物DCCT的制备方法,其特征在于,所述步骤2)中柱色谱分离的洗脱剂配比为乙酸乙酯:石油醚=1:6。
6.如权利要求1所述的荧光素衍生物DCCT在制备去甲肾上腺素检测试剂中的应用。
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