CN112939963B - 一种苯并吡喃酸酯衍生物及其合成方法和应用 - Google Patents
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Abstract
本发明提供了一种苯并吡喃酸酯衍生物及其合成方法和应用,所述衍生物的中文名称为7‑(二乙氨基)‑2‑(4‑(4‑(7‑((7‑硝基苯并[c][1,2,5]恶二唑‑4‑基)氧基)‑2‑氧‑2‑H‑色烯‑3‑羰基)哌嗪‑1‑基)苯基)盐,命名为PI‑LS2。本发明将PI‑LS2作为荧光探针,可实现三通道区分检测含硫化物。具体基于所述探针PI‑LS2,在PBS缓冲液(pH=7.4,含有20%CH3CN,v/v)溶液中通过紫外可见分光光度计和荧光光谱仪定量区分检测含硫化物。该方法可以对活细胞的线粒体和斑马鱼的硫醇和SO2进行专门成像。此外,该探针已成功应用于监测活细胞和斑马鱼的氧化应激和细胞凋亡过程。
Description
技术领域
本发明涉及以香豆素为母体的化合物,具体属于一种苯并吡喃酸酯衍生物及其合成方法,以及该衍生物在区分检测含硫化物中的应用。
背景技术
活性硫(RSS)主要包括三种生物硫醇,如高半胱氨酸(Hcy),半胱氨酸(Cys),谷胱甘肽(GSH)和二氧化硫(SO2)等。该化合物在细胞甚至生物体中无处不在,参与许多生理过程的调节,并且在维持生命系统的平衡中起着极其重要的作用。活性硫的浓度和代谢异常与许多疾病密切相关。病理研究表明,过量的亚硫酸盐/亚硫酸氢盐会在细胞和某些组织中引起毒性反应,并引起呼吸系统疾病,心血管疾病和神经系统疾病等。半胱氨酸(Cys)是细胞中硫代谢的中心,其缺乏与皮肤病,肝损害,智力低下等疾病有关。Hcy是在Cys生产过程中产生的中间产物,其浓度不平衡会导致疾病,例如神经系统疾病,心血管疾病,骨质疏松症和肠炎。GSH是细胞中最丰富的硫醇,GSH可以维持细胞中的氧化还原活性。当GSH浓度不平衡时,会引起其他疾病,例如阿尔茨海默氏病和肺部疾病等。因此,有必要开发一种新的区分检测方法。近年来,大量文献报道了荧光探针可检测其中一种活性硫。然而,大多数荧光探针难以有效地区分Cys,Hcy,GSH和SO2,许多探针的波长主要集中在短波区域。因此,开发用于检测生物活性硫的功能特异性近红外荧光探针具有重要意义。
众所周知,线粒体是细胞内RSS的主要来源,例如硫醇,并且在氧化还原稳态,药物代谢,信号转导以及其他生理和病理过程中起着至关重要的作用。有趣的是,带有荧光团的带正电荷的分子可以很好地靶向线粒体中的硫醇。更重要的是,我们使用该探针观察氧化应激和细胞凋亡期间的硫醇波动,证明它对于阐明生物体的病理生理过程非常有价值。这一发现为研究硫醇相关疾病的病理过程提供了有效的手段。
发明内容
本发明的目的是提供一种苯并吡喃酸酯衍生物及其合成方法,以及该衍生物可在区分检测含硫化物中应用。
本发明提供的一种苯并吡喃酸酯衍生物PI-LS2,所述衍生物的中文名称为7-(二乙氨基)-2-(4-(4-(7-((7-硝基苯并[c][1,2,5]恶二唑-4-基)氧基]-2-氧-2-H-色烯-3-羰基)哌嗪-1-基)苯基)盐,英文名称为7-(diethylamino)-2-(4-(4-(7-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)oxy)-2-oxo-2H-chromene-3-carbonyl)piperazin-1-yl)phenyl)chromenylium perchlorate。
其结构式为:
PI-LS2的合成方法,步骤为:
1)按摩尔比1:1将米氏酸和2,4-二羟基苯甲醛溶解在水中,混合物加热到100℃,回流2h后,自然冷却,有沉淀析出,抽滤即得米白色固体为化合物1。
2)按摩尔比1:1将4-哌嗪苯乙酮和4-(二乙氨基)-2-羟基苯甲醛缓慢加入浓硫酸中,在混合物加热到90℃保持8h,冷却至室温后,将反应混合物缓慢倒入冰水中,再加入HClO4搅拌,沉淀后的悬浮液用水洗净,真空干燥。产物经二氯甲烷-甲醇(v:v=15:1)柱层析纯化,得到紫黑色粉末即化合物2;
3)按摩尔比1:1:1将化合物1和1-乙基-3-(3-二甲基氨基丙基)碳二亚胺盐酸盐和1-羟基苯并三唑溶于无水N,N-二甲基甲酰胺中,混合物在氩气保护下0℃搅拌30min,然后按摩尔比1:0.2加入化合物2和三乙胺,混合物在室温下搅拌24h,反应完全后,将反应混合物倒入冰水中洗涤,抽滤得到黑色固体,产物经二氯甲烷-甲醇(v:v=15:1)柱层析纯化,得到深紫色化合物PI;
4)按摩尔比1:1:0.2将化合物PI,NBD-Cl和三乙胺溶于无水乙醇中,并将混合物在室温下搅拌过夜;产物经二氯甲烷-甲醇(v:v=15:1)柱层析纯化,得到深紫色化合物PI-LS2。
上述合成的PI-LS2可在细胞水平和斑马鱼中检测硫醇和二氧化硫(SO2)。
本发明提供的一种用苯并吡喃酸酯衍生物区分检测含硫化物的方法,包括如下步骤:
(1)、配制pH=7.4、浓度为10mM的PBS缓冲溶液,分别配制20mM的
(2)Hcy、GSH和Cys的水溶液,配制20mM的Na2SO3水溶液,配制2mM的PI-LS2的DMSO溶液;
(2)、取1600μL PBS缓冲溶液、400μL乙腈、10μL PI-LS2的DMSO溶液于比色皿中,在紫外可见光吸光光度计上监测探针PI-LS2随着Hcy,Cys的增加在475nm处的吸光度逐渐增加,但是随着GSH浓度的增加,吸光度没有变化;
(3)、取1600μL PBS缓冲溶液、400μL乙腈、10μL PI-LS2的DMSO溶液于比色皿中,在荧光分光光度仪上检测探针PI-LS2随着Hcy,Cys浓度的增大在550nm处的荧光强度逐渐增加,但是随着GSH浓度的增加,荧光强度基本没有变化。
(4)、取1600μL PBS缓冲溶液、400μL乙腈、10μL PI-LS2的DMSO溶液于比色皿中,在荧光分光光度仪上检测探针PI-LS2随着SO3 2-的增加在640nm处的荧光强度变化逐渐降低。
与现有技术相比,本发明的有益效果:
1、本发明PI-LS2是通过三个通道区分检测Cys,Hcy,GSH和SO2的双位点近红外荧光探针;
2、探针PI-LS2显示出对Cys,Hcy,GSH和SO2的快速响应时间;
3、探针PI-LS2可以对活细胞线粒体中的硫醇进行特殊成像;
4、探针PI-LS2可以观察到活细胞和斑马鱼在氧化应激和细胞凋亡过程中的硫醇通量。
附图说明
图1实施例1制备的PI-LS2的核磁氢谱
图2实施例1制备的PI-LS2的核磁碳谱
图3实施例1制备的PI-LS2的质谱图
图4 PI-LS2与硫醇作用的紫外光谱图
图5 PI-LS2与硫醇和SO3 2-作用的荧光光谱图
图6 PI-LS2与硫醇和SO3 2-在不同PH作用下的荧光强度图
图7 PI-LS2与各种分析物的荧光光谱图
图8 PI-LS2在不同细胞的线粒体定位成像
图9 PI-LS2测定加入硫醇和二氧化硫的细胞成像图
图10 PI-LS2建立炎症细胞模型的细胞成像图
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 PI-LS2的制备和表征:
PI-LS2的合成路线:
PI-LS2的合成方法,步骤为:
1)在50mL的圆底烧瓶中,将1g米氏酸和1g 2,4-二羟基苯甲醛溶解在20mL水中,混合物加热到100℃,回流2h后,自然冷却,有沉淀析出,抽滤即得米白色固体为化合物1。
2)在25mL的圆底烧瓶中,将408mg 4-哌嗪苯乙酮和386mg 4-(二乙氨基)-2-羟基苯甲醛缓慢加入10ml浓硫酸中,在混合物加热到90℃保持8h,冷却至室温后,将反应混合物缓慢倒入200mL冰水中,再加入1mL HClO4搅拌,沉淀后的悬浮液用水洗净,真空干燥。产物经二氯甲烷-甲醇(v:v=15:1)柱层析纯化,得到紫黑色粉末即化合物2;
3)在25mL的圆底烧瓶中,将103mg化合物1和380mg 1-乙基-3-(3-二甲基氨基丙基)碳二亚胺盐酸盐和270mg 1-羟基苯并三唑溶于15mL无水N,N-二甲基甲酰胺中,混合物在氩气保护下0℃搅拌30min,然后加入230mg化合物2和200μL三乙胺,混合物在室温下搅拌24h,反应完全后,将反应混合物倒入冰水中洗涤,抽滤得到黑色固体,产物经二氯甲烷-甲醇(v:v=15:1)柱层析纯化,得到深紫色化合物PI;
4)在25mL的圆底烧瓶中,将320mg化合物PI,290mg NBD-Cl和208μL三甲胺溶于10mL无水乙醇中,并将混合物在室温下搅拌过夜。产物经二氯甲烷-甲醇(v:v=15:1)柱层析纯化,得到深紫色化合物PI-LS2(0.10g,yield:30%).1H NMR(600MHz,DMSO-d6)δ8.73(d,J=8.1Hz,1H),8.65(d,J=8.4Hz,1H),8.36(s,1H),8.29(d,J=8.7Hz,2H),8.00(d,J=7.9Hz,2H),7.92(d,J=9.2Hz,1H),7.66(s,1H),7.50(d,J=8.4Hz,1H),7.37(d,J=9.0Hz,1H),7.31(s,1H),7.21(d,J=8.6Hz,2H),7.07(d,J=8.3Hz,1H),3.78(d,J=40.8Hz,4H),3.72-3.60(m,8H),1.25(s,7H).ESI-MS m/z:[M]+calcd for 713.2345;Found 713.2363(图1-3)。
实施例2
取2.0mL体积比为4:1的PBS和乙腈混合溶液、10μL浓度为2.0mmol/L的探针PI-LS2DMSO溶液合并于比色皿中,加入不同浓度的硫醇水溶液(0-100μM),15min后在紫外可见光吸光光度计上检测PI-LS2的吸光度变化。(图4)。随着Hcy,Cys的增加在475nm处的吸光度逐渐增加,但是随着GSH浓度的增加,吸光度没有变化。
实施例3
取2.0mL体积比为4:1的PBS和乙腈混合溶液、10μL浓度为2.0mmol/L的探针PI-LS2DMSO溶液合并于比色皿中,加入不同浓度的硫醇和SO3 2-水溶液(0-100μM),15min后在荧光光谱仪上测定探针PI-LS2在550nm和640nm处的荧光强度。随着Hcy,Cys的增加在550nm处的吸光度逐渐增加,但是随着GSH浓度的增加,吸光度没有变化。随着SO3 2-浓度的增加,荧光强度逐渐降低(图5)。
实施例4
配制不同pH、浓度为10mM的PBS缓冲溶液,配制2mM PI-LS2的DMSO溶液,配制20mM的硫醇水溶液,配制20mM的亚硫酸钠水溶液;在荧光比色皿中分别加入1600μL不同PH的PBS缓冲溶液、400μL乙腈和10μM PI-LS2的DMSO溶液,分别在荧光光谱仪上测定纯探针和探针加硫醇和SO3 2-的PH曲线,见图6。
实施例5
配制pH=7.4、浓度为10mM的PBS缓冲溶液,配制2mM PI-LS2的DMSO溶液,配制20mM的硫醇水溶液,配制20mM的亚硫酸钠水溶液;在荧光比色皿中,加1600μL PBS缓冲溶液、400μL乙腈和10μM PI-LS2的DMSO溶液,再分别加入100当量的其它分析物:L-Glu,L-Thr,L-Iso,L-Met,L-Val,L-Arg,L-Leu,L-Lys,L-Tyr,Hcy,GSH,Cys,在荧光分光光度仪上检测,绘制不同分析物对应的550nm处荧光强度柱状图(见图7)。Hcy,Cys使得检测体系在550nm处荧光强度明显升高,GSH处没有变化,亚硫酸钠使得检测体系在640nm处荧光强度明显降低,其它的分析物基本没有引起检测体系荧光强度的变化。
实施例6
将HeLa细胞,MCF-7细胞和HL-7702细胞中加入500μM Mito-Tracker Green,在37℃的恒温箱放置20min后,用PBS冲洗三次,再将探针PI-LS2的DMSO溶液加入上述三种细胞中,使得其浓度为10μM,放置20min后,用PBS冲洗细胞三次,再在荧光共聚焦显微镜下进行成像拍照(见图8)。红色通道为探针,绿色通道为Mito-Tracker Green,结果表明探针对线粒体具有良好的靶向性(皮尔逊系数0.90,0.86,0.87),说明探针能够特异性染色活细胞中的线粒体。
实施例7
配制pH=7.4、浓度为10mM的PBS缓冲溶液,配制2mM PI-LS2的DMSO溶液,配制20mM的硫醇水溶液,配制20mM的亚硫酸钠水溶液;先将HeLa细胞中加入2mL的PBS,再将探针溶液加入HeLa细胞中,使得其浓度为10μM,将HeLa细胞在37℃下放置20min后,用PBS冲洗细胞三次,再在荧光共聚焦显微镜下进行成像拍照,探针呈现三通道荧光。接着,将孵过探针的细胞加入10μM的硫醇,并在荧光共聚焦显微镜下观察荧光变化(见图9)。在加入Hcy和Cys时,体系在荧光成像仪下显示红色、蓝色和黄色通道荧光明显增强,而在加入GSH时,只有红色和蓝色通道荧光增强。而加入SO3 2-时,红色通道逐渐减弱。
实施例8
先将HeLa细胞中加入2mL的PBS,将药物BSO和PMA加入HeLa细胞中,使得其浓度为10μM,将HeLa细胞在37℃下放置20min后,用PBS冲洗细胞三次,再将探针PI-LS2溶液加入HeLa细胞中,使得其浓度为10μM,20min后在荧光共聚焦显微镜下观察荧光变化(见图10)。体系在荧光成像仪下显示红色通道荧光变暗。
上述实验结果说明PI-LS2是三通道区分检测线粒体中硫醇和二氧化硫变化的良好的候选者。
Claims (5)
2.如权利要求1所述的一种苯并吡喃酸酯衍生物PI-LS2的制备方法,其特征在于,包括如下步骤:
1)按摩尔比1:1将米氏酸和2,4-二羟基苯甲醛溶解在水中,混合物加热到100℃,回流2h后,自然冷却,有沉淀析出,抽滤即得米白色固体为化合物1;
2)按摩尔比1:1将4-哌嗪苯乙酮和4-(二乙氨基)-2-羟基苯甲醛缓慢加入浓硫酸中,在混合物加热到90℃保持8h,冷却至室温后,将反应混合物缓慢倒入冰水中,再加入HClO4搅拌,沉淀后的悬浮液用水洗净,真空干燥;产物经体积比15:1的二氯甲烷-甲醇柱层析纯化,得到紫黑色粉末即化合物2;
3)按摩尔比1:1:1将化合物1和1-乙基-3-(3-二甲基氨基丙基)碳二亚胺盐酸盐和1-羟基苯并三唑溶于无水N,N-二甲基甲酰胺中,混合物在氩气保护下0℃搅拌30min,然后按摩尔比1:0.2加入化合物2和三乙胺,混合物在室温下搅拌24h,反应完全后,将反应混合物倒入冰水中洗涤,抽滤得到黑色固体,产物经体积比15:1的二氯甲烷-甲醇柱层析纯化,得到深紫色化合物PI;
4)按摩尔比1:1:0.2将化合物PI,NBD-Cl和三乙胺溶于无水乙醇中,并将混合物在室温下搅拌过夜;产物经体积比15:1的二氯甲烷-甲醇柱层析纯化,得到深紫色化合物PI-LS2。
3.如权利要求1所述的PI-LS2在制备可区分检测含硫化物的探针中的应用。
4.一种非疾病诊断或治疗的用苯并吡喃酸酯衍生物区分检测含硫化物的方法,其特征在于,包括如下步骤:
(1)、配制pH=7.4、浓度为10mM的PBS缓冲溶液,分别配制20mM的Hcy、GSH和Cys的水溶液,配制20mM的Na2SO3水溶液,配制2mM的权利要求1所述PI-LS2的DMSO溶液;
(2)、取1600μL PBS缓冲溶液、400μL乙腈、10μL PI-LS2的DMSO溶液于比色皿中,在紫外可见光吸光光度计上监测探针PI-LS2随着Hcy、GSH、Cys的增加在475nm处的吸光度变化;
(3)、取1600μL PBS缓冲溶液、400μL乙腈、10μL PI-LS2的DMSO溶液于比色皿中,在荧光分光光度仪上检测探针PI-LS2随着Hcy、GSH和Cys的增加在550nm处的荧光强度变化;
(4)、取1600μL PBS缓冲溶液、400μL乙腈、10μL PI-LS2的DMSO溶液于比色皿中,在荧光分光光度仪上检测探针PI-LS2随着SO3 2-的增加在640nm处的荧光强度变化。
5.如权利要求1所述的PI-LS2在制备细胞成像试剂中的应用。
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