CN115322203A - 一种活细胞内质网自噬成像分析化合物及其制备方法 - Google Patents
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Abstract
Description
技术领域
本发明属于细胞生物学技术领域,具体涉及一种活细胞内质网自噬成像分析化合物及其制备方法。
背景技术
内质网(ER)是一种膜封闭的管状细胞器,介导蛋白质的加工、折叠和运输,以及代谢、离子储存等细胞活动。内质网稳态的破坏,与多种人类疾病相关[2-7]。因此发展内质网自噬的分析方法,对于探索内质网自噬相关的病理、生理作用[8]和开发内质网自噬诱导药物[5]具有重要意义。
迄今为止,内质网自噬主要通过检测ER蛋白的水平或者带有ER信号肽的荧光蛋白(如ssGFP-RFP-KDEL)。前者灵敏度较低,因为内质网自噬过程中消耗的蛋白数量远远小于内质网总体蛋白的数量,变化细微难以检测;而后者周期长,不适用于原代细胞。
发明内容
本发明目的在于克服现有技术缺陷,提供一种活细胞内质网自噬成像分析化合物。
本发明的另一目的在于提供上述活细胞内质网自噬成像分析化合物的制备方法。
本发明的技术方案如下:
一种活细胞内质网自噬成像分析化合物,其为荧光探针ER-proRed,其结构式为
上述活细胞内质网自噬成像分析化合物的制备方法,其合成路线如下:
在本发明的一个优选实施方案中,所述S1的合成包括:将ROX-EDA、丙炔酸和4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐加入DMF中,室温搅拌反应5-7h,然后依次经油泵抽干、复溶于DCM、饱和碳酸氢钠水溶液洗涤有机相、有机相脱水并减压浓缩和Al2O3柱层析纯化,即得所述S1。
进一步优选的,所述ROX-EDA、丙炔酸和4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐和DMF的比例为0.75mmol∶1.00mmol∶1.5mmol∶7.5mL。
在本发明的一个优选实施方案中,所述S3的合成包括:将所述S2、2-叠氮基乙氨、Pd(OAc)2、4,5-双二苯基膦-9,9-二甲基氧杂蒽和Cs2CO3加入无水甲苯中,于110-125℃加热回流0.8-1.2h,然后依次经减压浓缩、DCM复溶、水洗涤、萃取分离、有机相脱水并减压浓缩和硅胶柱层析分离纯化,即得所述S3。
进一步优选的,所述S2、2-叠氮基乙氨、Pd(OAc)2和4,5-双二苯基膦-9,9-二甲基氧杂蒽、Cs2CO3和无水甲苯的比例为0.31mmol∶0.48mmol∶0.31mmol∶0.047mmol∶0.94mmol∶10mL。
在本发明的一个优选实施方案中,所述ER-proRed的合成包括:将所述S1和所述S3溶解于四氢呋喃中,在氮气保护气氛下,加入预混的CuSO4·5H2O和抗坏血酸钠的混合水溶液,室温条件下反应8-15h,然后依次经减压浓缩、DCM复溶、饱和碳酸氢钠水溶液洗涤有机相、有机相脱水并减压浓缩和Al2O3柱层析纯化,即得所述ER-proRed。
进一步优选的,所述S1、所述S3和四氢呋喃的比例为0.13mmol∶0.20mmol∶2mL。
本发明的有益效果是:
1、如图1所示,在FHR的作用下,本发明在内质网内富集,发出绿色荧光;当发生内质网自噬时,本发明伴随着内质网进入酸性溶酶体,开启酸响应的强烈红色荧光信号,从而实现对内质网自噬过程的分析。
2、本发明合成工艺简单,易于推广。
3、本发明灵敏度高,适用性广,使用方便,可被用于细胞内质网自噬成像分析和筛选内质网自噬诱导剂等。
附图说明
图1为本发明的作用原理图。
图2为本发明实施例1中化合物ER-proRed的1H NMR(CDCl3)。
图3为本发明实施例1中化合物ER-proRed的13C NMR(CDCl3)。
图4为本发明实施例1中为化合物ER-proRed的高分辨质谱图。
图5为本发明实施例3中荧光探针ER-proRed对pH响应的发射光谱图。
图6为本发明实施例4中荧光探针ER-proRed在细胞中的内质网定位图。
图7为本发明实施例5中荧光探针ER-proRed用于饥饿诱导条件下的细胞内质网自噬成像分析结果图。
图8为本发明实施例6中荧光探针ER-proRed用于内质网自噬受体过表达条件下的细胞内质网自噬成像分析结果图。
具体实施方式
以下通过具体实施方式结合附图对本发明的技术方案进行进一步的说明和描述。
实施例1:制备荧光探针ER-proRed
本实施例制得的ER-proRed的结构式如下:
ER-proRed正常内质网中显示为绿色荧光信号,当发生内质网自噬时,探针ER-proRed伴随内质网进入溶酶体,在酸的响应下,发射出明亮的红色荧光信号。其合成路线如下:
具体包括如下步骤:
(1)ROX-EDA是根据Responsive hetero-organelle partition conferredfluorogenic sensing of mitochondrial depolarization.Chem Sci 2017;8:1915-21.中公开的合成方法得到的。向N,N-二甲基甲酰胺(DMF,7.5mL)中加入ROX-EDA(400.0mg,0.75mmol)、丙炔酸(70.0mg,1.00mmol)和4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐(DMTMM,4242.5mg,1.5mmol)。上述反应体系在室温下搅拌6小时,然后用油泵抽干。将剩余物溶解于二氯甲烷(DCM,20.0mL),用饱和碳酸氢钠水溶液洗涤有机相,然后分离出有机相并用无水硫酸钠脱水。浓缩有机相后,用Al2O3柱层析(洗脱剂:乙酸乙酯/石油醚,2:1)纯化得到化合物S1(50%,220.0mg)。
(2)化合物S2也是根据文献Synthesis of Fluorophores that Target SmallMolecules to the Endoplasmic Reticulum of Living Mammalian Cells.Angew ChemInt Ed Engl 2015;54:9696-9.中公开的合成方法得到的。在氩气保护条件下,向无水甲苯(10mL)中加入化合物S2(1400mg,0.31mmol),2-叠氮基乙氨(58mg,0.48mmol),Pd(OAc)2(7mg,0.031mmol),4,5-双二苯基膦-9,9-二甲基氧杂蒽(27.0mg,0.047mmol),Cs2CO3(305.0mg,0.94mmol)。在120℃条件下加热回流1小时。将烧瓶冷却至室温,减压浓缩,去除甲苯。将上述剩余物溶解于二氯甲烷(40mL),用水(40mL)洗涤,萃取分离。有机相用无水Na2SO4干燥,过滤,减压浓缩。所得粗产品用硅胶柱层析分离(洗脱剂:甲醇/二氯甲烷,1:50),得到橘红色固体化合物S3(57mg,45%)。
(3)将化合物S1(75.0mg,0.13mmol)和化合物S3(40mg,0.20mmol)溶解于四氢呋喃(2mL)中,并置于氮气保护条件。将CuSO4·5H2O(12mg,0.048mmol)和抗坏血酸钠(10mg,0.050mmol)分别溶解于超纯水(0.25mL),将二者迅速混合,并用注射器加入到上述反应体系中,室温条件下反应过夜,减压浓缩。将剩余物溶解在二氯甲烷(10.0mL)中,用25.0mL的饱和碳酸氢钠水溶液洗涤有机相,然后用无水硫酸钠干燥,过滤,减压浓缩。以二氯甲烷/甲醇(100:1)为洗脱剂,采用Al2O3柱层析纯化,得到产物如图2至图4所示的ER-proRed,产率为55%(54.0mg)。1H NMR(500MHz,CDCl3)δ8.37(s,1H),7.94-7.65(m,2H),7.40(ddt,J=29.8,14.8,4.9Hz,4H),7.16-7.02(m,2H),6.80(d,J=6.9Hz,1H),6.64-6.56(m,2H),6.03(d,J=20.9Hz,2H),4.78(t,J=5.9Hz,1H),3.96(q,J=6.0Hz,1H),3.38(t,J=6.0Hz,1H),3.20-2.98(m,8H),2.90(t,J=6.7Hz,4H),2.44(ddt,J=28.9,15.3,7.9Hz,3H),2.03(q,J=8.0,7.2Hz,6H),1.82(h,J=5.7Hz,4H),1.37(s,3H),1.27(d,J=14.6Hz,8H).13C NMR(126MHz,CDCl3)δ156.42,153.06,147.15,144.44,142.65,142.44,134.89,131.61,131.22,129.75,129.44,128.76,127.79,126.87,125.40,125.27,123.29,122.94,122.44,121.81,116.51,110.71,109.56,106.67,104.00,98.96,65.00,48.95,48.85,48.41,47.77,44.90,41.96,37.78,33.96,30.49,29.13,28.68,28.30,26.10,21.66,21.58,20.91,20.42,20.20,18.54,13.09.HRMS(ES+)calculated for C59H53F2N8O5 +(M+)m/z 991.4101,found:991.4123.
实施例2
配制浓度为10mM荧光探针ER-proRed的标准溶液:称取9.9mg的实施例1制得的ER-proRed溶于1mL二甲亚砜,即得10mmol/L(10mM)的荧光探针ER-proRed的标准溶液。
实施例3:ER-proRed对pH响应的荧光发射光谱。
取实施例2制备的荧光探针ER-proRed的标准溶液1μL稀释至1000μL(PBS,10mM,30%DMSO),得10μM的荧光探针ER-proRed溶液。取400μL上述溶液,检测荧光探针ER-proRed(10μM)在不同pH下(4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0),激发波长分别为495nm(FHR)和595nm(ROX)的荧光发射光谱。实验结果如图5,荧光探针ER-proRed对pH有响应。
实施例4
将ssRFP-KDEL+HeLa细胞(一种基因编辑的HeLa细胞,可表达内质网定位的RFP嵌合蛋白)播种于不同的35mm玻璃底培养皿中。第二天细胞贴壁后,将这些ssRFP-KDEL+细胞孵育5μM的荧光探针ER-proRed 60min。最后将上述细胞用PBS洗3次并进行共聚焦成像。结果如图6,探针ER-proRed很好地定位在内质网中。
实施例5
参照实施例4,HeLa细胞孵育5μM的荧光探针ER-proRed 60min,PBS洗三次后,加入LysoTracker Blue(一种溶酶体商业化探针)。随后细胞用PBS洗三次后加入HBSS(Hank′sBalanced Salt Solution)进行孵育6h,使细胞处于饥饿状态,对照组则用正常的全血清细胞培养液,同样经过6h。到时间点后进行共聚焦成像。结果如图7所示,饥饿处理过的HeLa细胞溶酶体的红光信号大大增强,说明本发明可用于指示内质网自噬的程度。
实施例6
参照实施例4,将野生型、HA-FAM134B+(一种基因编辑的HeLa细胞,可表达内质网自噬受体蛋白FAM134B)、HA-TEX264+(一种基因编辑的HeLa细胞,可表达内质网自噬受体蛋白TEX264)HeLa细胞播种于不同的35mm玻璃底培养皿中。第二天细胞贴壁后,将上述三种细胞分别孵育5μM的荧光探针ER-proRed 60min。然后将上述细胞用PBS洗3次,换上新鲜的细胞培养液培养6h,最后进行共聚焦成像。结果如图8所示,内质网自噬受体过表达的的HA-FAM134B+HeLa细胞和HA-TEX264+HeLa细胞中溶酶体的红光信号大大增强,再次说明本发明可用于指示内质网自噬,灵敏度高。
以上所述,仅为本发明的较佳实施例而已,故不能依此限定本发明实施的范围,即依本发明专利范围及说明书内容所作的等效变化与修饰,皆应仍属本发明涵盖的范围内。
Claims (10)
3.如权利要求2所述的制备方法,其特征在于:所述S1的合成包括:将ROX-EDA、丙炔酸和4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐加入DMF中,室温搅拌反应5-7h,然后依次经油泵抽干、复溶于DCM、饱和碳酸氢钠水溶液洗涤有机相、有机相脱水并减压浓缩和Al2O3柱层析纯化,即得所述S1。
4.如权利要求3所述的制备方法,其特征在于:所述ROX-EDA、丙炔酸和4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐和DMF的比例为0.75mmol∶1.00mmol∶1.5mmol∶7.5mL。
5.如权利要求2所述的制备方法,其特征在于:所述S3的合成包括:将所述S2、2-叠氮基乙氨、Pd(OAc)2、4,5-双二苯基膦-9,9-二甲基氧杂蒽和Cs2CO3加入无水甲苯中,于110-125℃加热回流0.8-1.2h,然后依次经减压浓缩、DCM复溶、水洗涤、萃取分离、有机相脱水并减压浓缩和硅胶柱层析分离纯化,即得所述S3。
6.如权利要求5所述的制备方法,其特征在于:所述S2、2-叠氮基乙氨、Pd(OAc)2和4,5-双二苯基膦-9,9-二甲基氧杂蒽、Cs2CO3和无水甲苯的比例为0.31mmol∶0.48mmol∶0.31mmol∶0.047mmol∶0.94mmol∶10mL。
7.如权利要求2所述的制备方法,其特征在于:所述ER-proRed的合成包括:将所述S1和所述S3溶解于四氢呋喃中,在氮气保护气氛下,加入预混的CuSO4·5H2O和抗坏血酸钠的混合水溶液,室温条件下反应8-15h,然后依次经减压浓缩、DCM复溶、饱和碳酸氢钠水溶液洗涤有机相、有机相脱水并减压浓缩和Al2O3柱层析纯化,即得所述ER-proRed。
8.如权利要求7所述的制备方法,其特征在于:所述S1、所述S3和四氢呋喃的比例为0.13mmol∶0.20mmol∶2mL。
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