CN106432164B - 一种香豆素衍生物docopa及其制备方法和应用 - Google Patents
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Abstract
本发明提供了一种香豆素衍生物DOCOPA及其制备方法和应用。所述的DOCOPA,英文名称为(E)‑4‑(3‑(7‑(diethylamino)‑2‑oxo‑2H‑chromen‑3‑yl)‑3‑oxoprop‑1‑en‑1‑yl)phenyl acrylate)。制备方法:先将3‑乙酰基‑7‑N,N‑二乙胺基香豆素、对羟基苯甲醛溶于乙腈中,加入催化量的哌啶,回流至反应完全;进一步得到中间化合物;再将中间化合物、三乙胺溶于二氯甲烷中,冰浴中逐渐滴加丙烯酰氯,室温搅拌至反应完全,减压蒸干后柱色谱分离得到DOCOPA。所述的应用,是基于DOCOPA在pH 7.8体系中对半胱氨酸和同型半胱氨酸区分检测,并用于细胞内半胱氨酸和同型半胱氨酸成像。DOCOPA对半胱氨酸和同型半胱氨酸显示了高的灵敏性和选择性。
Description
技术领域
本发明涉及检测硫醇检测分析技术,具体涉及一种香豆素衍生物DOCOPA及其制备方法,以及DOCOPA在检测半胱氨酸和同型半胱氨酸中的应用。
背景技术
半胱氨酸(Cys)是20种天然氨基酸中唯一具有还原性巯基(-SH)的氨基酸,它作为谷胱甘肽的前体,在生物体内扮演着氧化还原调节、生物催化等一系列重要的角色。同时,人体内非正常的Cys水平与儿童生长缓慢、肝损伤、皮肤损伤及肌肉损伤等疾病有着密切的关系。对生物体内的Cys进行检测具有非常重要的科学意义。
同型半胱氨酸(Hcy)是蛋氨酸和半胱氨酸代谢过程中一个重要的中间产物,它在体内主要通过再甲基化、转硫作用两种途径进行代谢。其代谢过程的异常将导致Hcy过度积累,增大患心血管和脑血管疾病的风险。目前,人体血浆总Hcy的含量已经成为临床预测心脏病、中风和老年痴呆症等疾病发病的主要指标。
由于Cys和Hcy结构和性质的相似性,使得荧光探针在对其进行区分检测标记时受到了极大限制,从而限制了其生理学和病理学作用的研究。因此开发能够用于Cys和Hcy区分检测的荧光探针受到人们的广泛关注。针对此现状,本专利利用具有优良荧光性质的香豆素衍生物作为荧光团、以丙烯酰基为识别位点,设计合成了探针DOCOPA((E)-4-(3-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-3-oxoprop-1-en-1-yl)phenyl acrylate)并用于Cys和Hcy的荧光区分检测。
发明内容
本发明的目的是提供一种香豆素衍生物及其制备方法,该衍生物可作为检测试剂用于Cys和Hcy的荧光区分检测;在区分检测Cys和Hcy时选择性高,响应速度快;该衍生物还可用于细胞内Cys和Hcy的荧光成像。
本发明提供的一种香豆素衍生物,名称为(E)-4-(3-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-3-oxoprop-1-en-1-yl)phenyl acrylate,缩写为DOCOPA,结构式为:
香豆素衍生物对Cys和Hcy显示了优良的灵敏性和选择性,检测过程简便、灵敏、快速。
DOCOPA的制备:
1)将3-乙酰基-7-N,N-二乙胺基香豆素、对羟基苯甲醛按摩尔比为1:1溶于乙腈中,加入催化量的哌啶,回流11小时至反应完全;体系冷却到室温后抽滤,乙腈洗涤,滤饼经干燥,得到化合物I。结构式如下:
2)将步骤1)所得化合物I、三乙胺溶于二氯甲烷中,冰浴中逐渐滴加丙烯酰氯,滴加完毕后室温搅拌20小时至反应完全;体系减压蒸干后柱色谱分离(按体积比乙酸乙酯:石油醚=1:3)得到目标化合物DOCOPA。化合物I、三乙胺和丙烯酰氯的摩尔比为1:1:1.4。
DOCOPA的合成路线如下:
本发明香豆素衍生物DOCOPA的用途:该衍生物可以在pH 7.8体系中对水环境和生物细胞体系中半胱氨酸和同型半胱氨酸的区分检测;所述的检测包含荧光检测和细胞成像检测。
与现有技术相比,本发明具有如下有益效果:
1、所述香豆素衍生物合成简单,成本低廉,易于大规模生产;
2、所述检测方法能够实现Cys和Hcy的区分检测,其它氨基酸不干扰对Cys和Hcy的测定;
3、所述检测方法可用于细胞内Cys和Hcy的分别成像,其在Cys和Hcy生理学和病理学研究中具有广阔的应用前景;
4、检测手段简单,只需要借助荧光分光光度仪和激光共聚焦显微镜即可实现。
附图说明:
图1DOCOPA氢谱表征
图2DOCOPA碳谱表征
图3DOCOPA质谱表征
图4实施例2DOCOPA与Cys作用0-12min内的荧光发射图
图5实施例2DOCOPA与Cys作用14-120min内的荧光发射图
图6实施例3DOCOPA与Hcy作用的荧光发射图
图7实施例4DOCOPA与各种分析物作用的荧光发射
图8实施例5DOCOPA对细胞外源Cys和Hcy进行区分成像
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
DOCOPA的制备:
1)化合物I的合成:
取3-乙酰基-7-N,N-二乙胺基香豆素(6mmol,1.55g)、对羟基苯甲醛(6mmol,0.732g)溶于30mL乙腈中,加入3~5滴哌啶,回流11小时至反应完全;体系冷却到室温后抽滤,乙腈洗涤,滤饼经干燥,得到化合物I。
2)化合物DOCOPA的合成:
取化合物I(1mmol,0.36g)、三乙胺(1mmol)溶于二氯甲烷中,冰浴中逐渐滴加丙烯酰氯(1.4mmol),滴加完毕后室温搅拌20小时至反应完全;体系减压蒸干后柱色谱分离(按体积比乙酸乙酯:石油醚=1:3)得到目标化合物DOCOPA。
1H NMR(600MHz,DMSO-d6):δ8.62(s,1H),7.96(d,J=15.8Hz,1H),7.80(d,J=8.4Hz,2H),7.75–7.66(m,2H),7.30(d,J=8.5Hz,2H),6.83(d,J=9.0Hz,1H),6.63(s,1H),6.57(d,J=17.4Hz,1H),6.44(dd,J=17.3,10.4Hz,1H),6.19(d,J=9.7Hz,1H),3.52(dd,J=13.2,6.3Hz,4H),1.16(t,J=6.9Hz,6H)(图1)。13C NMR(151MHz,DMSO-d6):δ186.0,164.4,160.4,158.8,153.5,152.1,149.0,141.3,134.4,133.3,132.9,130.1,128.0,125.8,122.9,115.9,110.8,108.4,96.4,45.0,12.9(图2)。HR MS[M+H]+:m/z Calcd418.1649,Found 418.1653(图3)。
实施例2
配制pH=7.8、浓度为10mM的HEPES缓冲溶液,配制2mM DOCOPA的DMSO溶液,配制20mM Cys的水溶液;取2mL的HEPES/DMSO(v/v,1:1,pH 7.8)溶液、30μL DOCOPA的DMSO溶液加到一个荧光比色皿中,加入30μL Cys的水溶液,随时间在荧光分光光度仪上检测(447nm激发)。0-12min内,499nm的荧光强度逐渐增强;12min以后,499nm的荧光强度逐渐降低,554nm的荧光强度逐渐增强,荧光发射图见图4和图5。
实施例3
配制pH=7.8、浓度为10mM的HEPES缓冲溶液,配制2mM DOCOPA的DMSO溶液,配制20mM Hcy的水溶液;取2mL的HEPES/DMSO(v/v,1:1,pH 7.8)溶液、30μL DOCOPA的DMSO溶液加到一个荧光比色皿中,加入30μL Hcy的水溶液,随时间在荧光分光光度仪上检测(447nm激发)。499nm的荧光强度逐渐增强,荧光发射图见图6。
实施例4
配制pH=7.8、浓度为10mM的HEPES缓冲溶液,配制2mM DOCOPA的DMSO溶液,分别配制20mM Cys,Hcy,GSH,Ala,Asn,Arg,Asp,Gln,Glu,Gly,His,Ile,Leu,Lys,Met,Phe,Pro,Ser,Thr,Trp,Tyr和Val的水溶液;在22个荧光比色皿中,各加入2mL的HEPES/DMSO(v/v,1:1,pH 7.8)溶液、30μL DOCOPA的DMSO溶液,再分别加入10摩尔当量的Cys,Hcy,GSH,Ala,Asn,Arg,Asp,Gln,Glu,Gly,His,Ile,Leu,Lys,Met,Phe,Pro,Ser,Thr,Trp,Tyr和Val。2h后在荧光分光光度仪上检测(447nm激发),(见图7)。Cys使得检测体系在554nm处荧光强度明显升高,同时,Hcy使得检测体系在499nm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Cys和Hcy的区分检测。
实施例5
配置4mM DOCOPA的DMSO溶液,配置4mM尼日利亚菌素的DMSO溶液,分别配制20mMCys和Hcy的水溶液,配制200mM N-乙基马来酰亚胺的DMSO溶液;在三个育有HepG2细胞的培养皿中分别加入10μL N-乙基马来酰亚胺的DMSO溶液,并对其进行编号。30min后,弃掉培养液,用PBS缓冲溶液洗涤。在以上三个培养皿中分别加入2mL、pH 7.8的HBSS缓冲液,再分别加入5μL尼日利亚菌素的DMSO溶液和5μL DOCOPA的DMSO溶液培养。30min后,弃掉培养液,用pH 7.8的HBSS缓冲液洗涤。一号培养皿作为对照组,加入2mL、pH 7.8的HBSS缓冲液和5μL尼日利亚菌素的DMSO溶液;二号培养皿加入2mL、pH 7.8的HBSS缓冲液、5μL尼日利亚菌素的DMSO溶液和10μL Cys的水溶液;三号培养皿加入2mL、pH 7.8的HBSS缓冲液、5μL尼日利亚菌素的DMSO溶液和10μL Hcy的水溶液培养。60min后,弃掉培养液,用pH 7.8的HBSS缓冲液洗涤,用激光共聚焦显微镜进行成像。激发:458nm;红色通道:600±25nm;绿色通道:500±20nm。如图8,一号培养皿在绿色通道和红色通道下均无荧光发射(a,d),二号培养皿在绿色通道和红色通道均表现为弱荧光发射(b,e),三号培养皿绿色通道变现强荧光发射(c),而红色通道无荧光发射(f)。因此,通过分别检测绿色通道和红色通道的荧光发射可以实现化合物DOCOPA对细胞内Cys和Hcy的区分成像。
Claims (6)
1.一种香豆素衍生物DOCOPA,其特征在于,结构式为:
2.如权利要求1所述的一种香豆素衍生物DOCOPA的制备方法,其特征在于,包括如下步骤:
1)将3-乙酰基-7-N,N-二乙胺基香豆素、对羟基苯甲醛溶于乙腈中,加入催化量的哌啶,回流至反应完全;体系冷却到室温后抽滤,乙腈洗涤,滤饼经干燥,得到中间体化合物I,结构式如下:
2)将步骤1)所得化合物I、三乙胺溶于二氯甲烷中,冰浴中逐渐滴加丙烯酰氯,室温搅拌至反应完全;体系减压蒸干后柱色谱分离得到目标化合物DOCOPA。
3.如权利要求2所述的香豆素衍生物DOCOPA的制备方法,其特征在于,所述步骤1)中3-乙酰基-7-N,N-二乙胺基香豆素和对羟基苯甲醛的摩尔比为1:1。
4.如权利要求2所述的香豆素衍生物DOCOPA的制备方法,其特征在于,所述步骤2)中化合物I、三乙胺和丙烯酰氯的摩尔比为1:1:1.4,柱色谱的洗脱剂配比为乙酸乙酯:石油醚=1:3。
5.如权利要求1所述的香豆素衍生物DOCOPA在制备半胱氨酸或同型半胱氨酸检测试剂中的应用。
6.如权利要求1所述的香豆素衍生物DOCOPA在制备区分半胱氨酸和同型半胱氨酸的检测试剂中的应用。
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