CN111334074A - 一种高亮度、高稳定性线粒体荧光染料 - Google Patents
一种高亮度、高稳定性线粒体荧光染料 Download PDFInfo
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Abstract
本发明提供了一种高亮度、高稳定性线粒体荧光染料。该染料为以萘酰亚胺为染料母体,以三苯基膦为定位基团设计合成的一种能够对活细胞线粒体快速、准确标记的荧光染料。通过在供电子一端引入氮杂环丁烷,一定程度上抑制了染料的TICT,使其亮度和稳定性大幅度提高,水中吸光度可达16000M‑1cm‑1,量子产率为0.27,稳定性明显增加,适用于线粒体的超分辨显微成像,在生物成像领域具有广泛的应用前景。
Description
技术领域
本发明属于荧光染料和成像领域,具体涉及一种高亮度、高稳定性线粒体荧光染料。
背景技术
线粒体被认为是细胞内最重要的细胞器之一,监测其形态及与多种细胞器的相互作用,对深刻理解复杂的细胞生命活动非常必要。光学显微镜因其原位无损的检测特点,在这类生命科学领域的研究中备受青睐。尤其是近些年来发展起来的超分辨显微成像技术,其高于20nm的分辨率为细胞器水平的研究带来了新机遇。而超分辨显微成像技术所使用的高功率激光器,对细胞器定位荧光染料的亮度和稳定性提出了更高要求。
目前广泛使用的绿色商业线粒体染料为基于花菁染料的MitoTracker Green,花菁染料易被生物体内的单线态氧氧化而淬灭,光稳定性差、量子产率低,极大地限制了超分辨显微成像技术在线粒体相关研究领域应用。因此,开发一种高亮度、高稳定性荧光染料对于活细胞线粒体超分辨荧光成像显得尤为迫切。
发明内容
本发明提供了一种高亮度、高稳定性线粒体荧光染料,,该染料克服了商业线粒体荧光染料亮度、光稳定性差的缺点。具体结构以1,8-萘酰亚胺为荧光团,以三苯基膦为靶向基团,借助线粒体自身显负性的膜电位即可实现对线粒体的定位,从而实现对线粒体超分辨显微像。
本发明一种高亮度、高稳定性线粒体荧光染料,其结构式如下所示:
一种高亮度、高稳定性线粒体荧光染料的合成方法,合成步骤如下:
合成路线:
具体合成方法如下:
(1)中间体N-(6-羟基己基)-4-溴-1,8-萘酐的合成
将4-溴-1,8-萘酐和6-氨基-1-己醇混合于乙醇中,加热至60-90℃,搅拌24-48h。反应结束后减压除去溶剂,硅胶色谱柱分离得到N-(6-羟基己基)-4-溴-1,8-萘酐。
(2)中间体N-(6-溴己基)-4-溴-1,8-萘酐的合成
将N-(6-羟基己基)-4-溴-1,8-萘酐溶于乙酸乙酯中,向其中滴加0.5-1.5g三溴化磷,缓慢升温至50-70℃搅拌6-8h,反应结束后减压除去溶剂,硅胶色谱柱分离得到N-(6-溴己基)-4-溴-1,8-萘酐。
(3)中间体N-(6-三苯基膦己基)-4-溴-1,8-萘酐的合成
将N-(6-溴己基)-4-溴-1,8-萘酐和三苯基膦混合于乙腈中,密封管中升温至120-140℃搅拌8-16h,反应结束后减压除去溶剂,硅胶色谱柱分离得到N-(6-三苯基膦己基)-4-溴-1,8-萘酐。
(4)化合物Mito-Aze的合成
将N-(6-三苯基膦己基)-4-溴-1,8-萘酐溶于乙二醇甲醚中,向其中滴加氮杂环丁烷,升温至120-140℃搅拌8-16h,反应结束后减压除去溶剂,硅胶色谱柱分离得到化合物Mito-Aze。
步骤(1)中:4-溴-1,8-萘酐与6-氨基-1-己醇的质量比为1:0.3-5,4-溴-1,8-萘酐的质量与乙醇的体积比为1:3-20g/mL。
步骤(2)中:N-(6-羟基己基)-4-溴-1,8-萘酐与三溴化磷的质量比为1:0.25-1.5,N-(6-羟基己基)-4-溴-1,8-萘酐的质量与乙酸乙酯的体积比为1:5-10g/mL。
步骤(3)中:N-(6-溴己基)-4-溴-1,8-萘酐与三苯基膦的质量比为1:0.5-3,N-(6-溴己基)-4-溴-1,8-萘酐的质量与乙腈的体积比为1:5-20g/mL
步骤(4)中:N-(6-三苯基膦己基)-4-溴-1,8-萘酐与氮杂环丁烷的质量比为1-16:1,N-(6-三苯基膦己基)-4-溴-1,8-萘酐的质量与乙二醇甲醚的体积比为1:10-50g/mL。
一种高亮度、高稳定性线粒体荧光染料在荧光成像、分子探针及荧光传感领域的应用。
本发明具有以下特征:
该探针原料廉价易得,合成方法简单等优点。
该探针在水中的量子产率达到0.27,吸光度达到16000M-1cm-1,相比于常规萘酰亚胺染料亮度明显提高,在HeLa、MCF等细胞中都能实现对线粒体的准确定位,适用于线粒体的超分辨显微成像。
附图说明
图1为实施例1制备得到的化合物Mito-Aze的核磁氢谱;
图2为实施例1制备得到的化合物Mito-Aze的核磁碳谱;
图3为实施例1制备得到的化合物Mito-Aze的高分辨质谱;
图4为实施例1制备得到的化合物Mito-Aze在不同溶剂中的归一化荧光谱图,横坐标为波长,纵坐标为荧光强度,荧光探针的浓度为10μM;
图5为实施例1制备得到的化合物Mito-Aze在不同溶剂中的归一化紫外吸收谱图,横坐标为波长,纵坐标为吸收强度,荧光探针的浓度为10μM;
图6为实施例1制备的探针Mito-Aze与荧光素在PBS缓冲液(20mM,pH=7.4)中,经过不同时间激光照射后后其最大荧光发射强度的变化,横坐标为激光照射时间,纵坐标为相对荧光强度,即最大荧光发射强度与初始最大荧光发射强度的比值;
图7为实施例1中制备得到的化合物Mito-Aze标记的宫颈癌细胞(HeLa)线粒体共聚焦成像图;
图8为实施例1中制备得到的化合物Mito-Aze标记的HeLa细胞线粒体结构光照明显微成像图。
具体实施方式
实施例1.
线粒体探针Mito-Aze的合成。
中间体N-(6-羟基己基)-4-溴-1,8-萘酐的合成
4-溴-1,8-萘酐(0.50g,1.81mmol)和6-氨基-1-己醇(0.64g,5.44mmol)混合于10mL乙醇中,升温至80℃,反应结束8h后减压除去溶剂,残余物经硅胶柱(二氯甲烷/甲醇=80/1,V/V)分离得灰白色固体0.58g,产率86%。
其高分辨质谱数据如下:
高分辨质谱C18H18BrNO3 +[M]+计算值:376.0548;实验值:376.0521。
经检测,其产物结构为N-(6-羟基己基)-4-溴-1,8-萘酐。
中间体N-(6-溴己基)-4-溴-1,8-萘酐的合成
N-(6-羟基己基)-4-溴-1,8-萘酐(0.30g,0.80mmol)溶于10mL乙酸乙酯中,向其中滴加三溴化磷(0.64g,2.4mmol),缓慢升温至70℃搅拌6h,减压出去溶剂,残余物经硅胶柱(二氯甲烷/甲醇=100/1,V/V)分离得灰白色固体0.22g,产率63%。
其核磁谱图氢谱数据如下:
1H NMR(400MHz,CDCl3)δ8.65(dd,J=7.3,0.9Hz,1H),8.57(dd,J=8.5,0.8Hz,1H),8.41(d,J=7.9Hz,1H),8.04(d,J=7.9Hz,1H),7.85(dd,J=8.4,7.4Hz,1H),4.23–4.03(m,2H),3.41(t,J=6.8Hz,2H),1.95–1.82(m,2H),1.81–1.66(m,2H),1.58–1.34(m,4H).
其核磁谱图碳谱数据如下:
13C NMR(101MHz,CDCl3)δ163.64,163.62,133.30,132.07,131.26,131.12,130.64,130.29,129.01,128.10,123.09,122.22,40.39,33.83,32.67,29.72,27.87,26.26.
经检测,其产物结构为N-(6-溴己基)-4-溴-1,8-萘酐。
中间体N-(6-三苯基膦己基)-4-溴-1,8-萘酐的合成
N-(6-溴己基)-4-溴-1,8-萘酐(0.30g,0.69mmol)和三苯基膦(0.91g,3.45mmol)混合于5mL乙腈中,密封管中升温至140℃搅拌10h,反应结束后减压除去溶剂,残余物经硅胶柱(二氯甲烷/甲醇=100/1,V/V)分离得白色固体0.39g,产率82%。
其核磁谱图氢谱数据如下:
1H NMR(400MHz,CDCl3)δ8.55(d,J=7.3Hz,1H),8.51(d,J=8.5Hz,1H),8.31(d,J=7.9Hz,1H),7.99(d,J=7.9Hz,1H),7.82(ddd,J=12.8,10.1,7.6Hz,10H),7.71(td,J=7.6,3.4Hz,6H),4.07(t,J=7.3Hz,2H),3.78–3.68(m,2H),1.70–1.60(m,4H),1.45–1.36(m,2H).
经检测,其产物结构为N-(6-三苯基膦己基)-4-溴-1,8-萘酐。
线粒体探针Mito-Aze的合成
将N-(6-三苯基膦己基)-4-溴-1,8-萘酐(0.1g,0.14mmol)溶于5mL乙二醇甲醚中,向其中滴加氮杂环丁烷(42mg,0.72mmol),升温至120℃搅拌10h,反应结束后减压除去溶剂,残余物经硅胶柱(二氯甲烷/甲醇=50/1,V/V)分离得橙色固体33mg,产率40%。
其核磁谱图氢谱如下图1所示,具体数据如下:
1H NMR(400MHz,CDCl3)δ8.48(d,J=7.2Hz,1H),8.33(d,J=8.1Hz,1H),8.26(d,J=8.4Hz,1H),7.80(dt,J=14.3,7.3Hz,9H),7.71(dd,J=7.1,2.7Hz,6H),7.51(t,J=7.8Hz,1H),6.38(d,J=8.4Hz,1H),4.51(t,J=7.4Hz,4H),4.06(t,J=7.0Hz,2H),3.69(s,2H),2.65–2.54(m,2H),1.80(s,2H),1.67(d,J=11.6Hz,4H),1.39(s,2H).
其核磁谱图碳谱如下图2所示,具体数据如下:
13C NMR(101MHz,CDCl3)δ164.72,164.04,152.58,135.05,133.72,133.62,133.28,131.09,130.60,130.48,130.25,123.74,118.71,117.85,116.16,106.23,55.44,29.70,27.47,26.35,22.29,17.10.
其高分辨质谱谱图如下图3所示,具体数据如下:
高分辨质谱计算值C39H38N2O2P+[M]+:1054.4134;实验值:1054.4212。
经检测,其产物结构如上式Mito-Aze所示,能够准确定位于活细胞线粒体,亮度和稳定性满足长时间荧光成像的需求,其性能如下:
将该探针溶解于DMSO溶液中,配制成2mM母液,根据需要配制成不同浓度测试溶液,以检测其荧光光谱变化及细胞内线粒体荧光成像。
Mito-Aze在乙腈、氯仿、乙醇、二甲基亚砜、水中的荧光发射及紫外吸收光谱测试。每次取20μL荧光染料母液,分别加入4mL乙腈、氯仿、乙醇、二甲基亚砜、水,配制成10μM的荧光染料测试液,分别进行荧光发射和紫外吸收光谱的测试。
Mito-Aze在乙腈、氯仿、乙醇、二甲基亚砜、水中的归一化荧光发射谱图如图4所示;Mito-Aze在乙腈、氯仿、乙醇、二甲基亚砜、水中的归一化紫外吸收谱图如图5所示;其中Mito-Aze的浓度为10μM,该探针在水中的量子产率达到0.27,吸光度达到16000M-1cm-1,相比于常规萘酰亚胺染料亮度明显提高,具有很高的亮度。
Mito-Aze与荧光素在PBS缓冲液(20mM,pH=7.4)中的光稳定性测试。取20μLMito-Aze、荧光素母液,分别加入4mL PBS缓冲液中,配制成10μM的荧光探针测试液,并在500W钨灯下连续照射。光源距离样品50cm,每次将测试液温度稳定在25℃后进行荧光光谱测试。采取0,0.5,1,1.5,2,3,4,5,6,8,10h为时间点分别测试。
Mito-Aze与荧光素在不同时间激光照射后,最大荧光发射强度与初始最大荧光发射强度的比值如图6所示;其中荧光探针浓度均为10μM,在连续照射10h后,Mito-Aze的荧光发射强度相比初始最大荧光发射强度仅下降了约10%,而荧光素染料的发射强度下降了近90%,证明Mito-Aze有很好的光稳定性。
实施例2.
线粒体探针Mito-Aze的合成。
中间体N-(6-羟基己基)-4-溴-1,8-萘酐的合成
4-溴-1,8-萘酐(0.50g,1.81mmol)和6-氨基-1-己醇(0.3g,2.72mmol)混合于10mL乙醇中,升温至80℃,反应结束8h后减压除去溶剂,残余物经硅胶柱(二氯甲烷/甲醇=80/1,V/V)分离得灰白色固体0.47g,产率67%。
中间体N-(6-溴己基)-4-溴-1,8-萘酐的合成
N-(6-羟基己基)-4-溴-1,8-萘酐(0.30g,0.80mmol)溶于10mL乙酸乙酯中,向其中滴加三溴化磷(0.15g,0.61mmol),缓慢升温至60℃搅拌6h,减压出去溶剂,残余物经硅胶柱(二氯甲烷/甲醇=100/1,V/V)分离得灰白色固体0.14g,产率56%。
中间体N-(6-三苯基膦己基)-4-溴-1,8-萘酐的合成
N-(6-溴己基)-4-溴-1,8-萘酐(0.30g,0.69mmol)和三苯基膦(0.15g,0.57mmol)混合于5mL乙腈中,密封管中升温至120℃搅拌10h,反应结束后减压除去溶剂,残余物经硅胶柱(二氯甲烷/甲醇=100/1,V/V)分离得白色固体0.26g,产率69%。。
线粒体探针Mito-Aze的合成
将N-(6-三苯基膦己基)-4-溴-1,8-萘酐(0.1g,0.14mmol)溶于5mL乙二醇甲醚中,向其中滴加氮杂环丁烷(6mg,0.12mmol),升温至120℃搅拌10h,反应结束后减压除去溶剂,残余物经硅胶柱(二氯甲烷/甲醇=50/1,V/V)分离得橙色固体21mg,产率30%。
经检测,其产物结构如上式Mito-Aze所示,其在水中荧光发射波长为560nm,紫外吸收为470nm。
实施例3.
线粒体探针Mito-Aze的合成。
中间体N-(6-羟基己基)-4-溴-1,8-萘酐的合成
4-溴-1,8-萘酐(0.50g,1.81mmol)和6-氨基-1-己醇(2.5g,22mmol)混合于10mL乙醇中,升温至80℃,反应结束8h后减压除去溶剂,残余物经硅胶柱(二氯甲烷/甲醇=80/1,V/V)分离得灰白色固体0.64g,产率92%。
中间体N-(6-溴己基)-4-溴-1,8-萘酐的合成
N-(6-羟基己基)-4-溴-1,8-萘酐(0.30g,0.80mmol)溶于10mL乙酸乙酯中,向其中滴加三溴化磷(0.45g,1.83mmol),缓慢升温至60℃搅拌8h,减压出去溶剂,残余物经硅胶柱(二氯甲烷/甲醇=100/1,V/V)分离得灰白色固体0.19g,产率67%。
中间体N-(6-三苯基膦己基)-4-溴-1,8-萘酐的合成
N-(6-溴己基)-4-溴-1,8-萘酐(0.30g,0.69mmol)和三苯基膦(0.15g,0.57mmol)混合于5mL乙腈中,密封管中升温至120℃搅拌10h,反应结束后减压除去溶剂,残余物经硅胶柱(二氯甲烷/甲醇=100/1,V/V)分离得白色固体0.26g,产率69%。
线粒体探针Mito-Aze的合成
将N-(6-三苯基膦己基)-4-溴-1,8-萘酐(0.1g,0.14mmol)溶于5mL乙二醇甲醚中,向其中滴加氮杂环丁烷(0.1g,2mmol),升温至140℃搅拌10h,反应结束后减压除去溶剂,残余物经硅胶柱(二氯甲烷/甲醇=50/1,V/V)分离得橙色固体38mg,产率45%。
经检测,其产物结构如上式Mito-Aze所示,其在水中荧光发射波长为560nm,紫外吸收为470nm。
实施例4.
Mito-Aze对活细胞染色实验。取0.5μL母液溶于1mL细胞培养液中,37℃,5%CO2下孵育30分钟后分别进行荧光共聚焦成像及结构光照明显微成像实验。
Mito-Aze终浓度为1μM的细胞培养液孵育宫颈癌细胞(HeLa细胞)10分钟后共聚焦荧光成像图如图7所示,HeLa细胞内线形的线粒体清晰可见。
Mito-Aze终浓度为1μM的细胞培养液孵育宫颈癌细胞(HeLa细胞)10分钟后结构光照明显微成像图如图8所示,HeLa细胞内线形的线粒体清晰可见,Mito-Aze能够用于活细胞内线粒体标记。
Claims (7)
2.如权利要求1所述一种高亮度、高稳定性线粒体荧光染料的合成方法,其特征在于:该合成的具体步骤如下:
(1)中间体N-(6-羟基己基)-4-溴-1,8-萘酐的合成
将4-溴-1,8-萘酐和6-氨基-1-己醇混合于乙醇中,加热至60-90℃,搅拌24-48h;反应结束后减压除去溶剂,硅胶色谱柱分离得到N-(6-羟基己基)-4-溴-1,8-萘酐;
(2)中间体N-(6-溴己基)-4-溴-1,8-萘酐的合成
将N-(6-羟基己基)-4-溴-1,8-萘酐溶于乙酸乙酯中,向其中滴加0.5-1.5g三溴化磷,缓慢升温至50-70℃搅拌6-8h,反应结束后减压除去溶剂,硅胶色谱柱分离得到N-(6-溴己基)-4-溴-1,8-萘酐;
(3)中间体N-(6-三苯基膦己基)-4-溴-1,8-萘酐的合成
将N-(6-溴己基)-4-溴-1,8-萘酐和三苯基膦混合于乙腈中,密封管中升温至120-140℃搅拌8-16h,反应结束后减压除去溶剂,硅胶色谱柱分离得到N-(6-三苯基膦己基)-4-溴-1,8-萘酐;
(4)化合物Mito-Aze的合成
将N-(6-三苯基膦己基)-4-溴-1,8-萘酐溶于乙二醇甲醚中,向其中滴加氮杂环丁烷,升温至120-140℃搅拌8-16h,反应结束后减压除去溶剂,硅胶色谱柱分离得到化合物Mito-Aze。
3.如权利要求2中所述的一种高亮度、高稳定性线粒体荧光染料的合成方法,其特征在于:步骤(1)中,4-溴-1,8-萘酐与6-氨基-1-己醇的质量比为1:0.3-5,
4-溴-1,8-萘酐的质量与乙醇的体积比为1:3-20g/mL。
4.如权利要求2中所述的一种高亮度、高稳定性线粒体荧光染料的合成方法,其特征在于:步骤(2)中,N-(6-羟基己基)-4-溴-1,8-萘酐与三溴化磷的质量比为1:0.25-1.5,
N-(6-羟基己基)-4-溴-1,8-萘酐的质量与乙酸乙酯的体积比为1:5-10g/mL。
5.如权利要求2中所述的一种高亮度、高稳定性线粒体荧光染料的合成方法,其特征在于:步骤(3)中,N-(6-溴己基)-4-溴-1,8-萘酐与三苯基膦的质量比为1:0.5-3,
N-(6-溴己基)-4-溴-1,8-萘酐的质量与乙腈的体积比为1:5-20g/mL。
6.如权利要求2中所述一种高亮度、高稳定性线粒体荧光染料的合成方法,其特征在于:步骤(4)中,N-(6-三苯基膦己基)-4-溴-1,8-萘酐与氮杂环丁烷的质量比为1-16:1,
N-(6-三苯基膦己基)-4-溴-1,8-萘酐的质量与乙二醇甲醚的体积比为1:10-50g/mL。
7.如权利要求1所述的一种高亮度、高稳定性线粒体荧光染料在荧光成像、分子探针及荧光传感领域的应用。
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112300213A (zh) * | 2020-10-29 | 2021-02-02 | 赤峰学院 | 固态/溶液中可逆变色可靶向线粒体的荧光染料及制备方法和应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105777637A (zh) * | 2014-12-22 | 2016-07-20 | 中国科学院化学研究所 | 一种水溶性线粒体靶向成像探针及其制备方法 |
CN107603269A (zh) * | 2016-07-11 | 2018-01-19 | 华东理工大学 | 一类基于萘酰亚胺的荧光染料、其制备方法及应用 |
CN108069902A (zh) * | 2016-11-14 | 2018-05-25 | 中国科学院大连化学物理研究所 | 一类标记和/或检测细胞中脂滴的荧光探针及其制备和应用 |
CN108276442A (zh) * | 2018-03-08 | 2018-07-13 | 济南大学 | 一种线粒体靶向甲醛荧光探针及其制备方法和应用 |
-
2018
- 2018-12-18 CN CN201811549738.8A patent/CN111334074A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105777637A (zh) * | 2014-12-22 | 2016-07-20 | 中国科学院化学研究所 | 一种水溶性线粒体靶向成像探针及其制备方法 |
CN107603269A (zh) * | 2016-07-11 | 2018-01-19 | 华东理工大学 | 一类基于萘酰亚胺的荧光染料、其制备方法及应用 |
CN108069902A (zh) * | 2016-11-14 | 2018-05-25 | 中国科学院大连化学物理研究所 | 一类标记和/或检测细胞中脂滴的荧光探针及其制备和应用 |
CN108276442A (zh) * | 2018-03-08 | 2018-07-13 | 济南大学 | 一种线粒体靶向甲醛荧光探针及其制备方法和应用 |
Non-Patent Citations (1)
Title |
---|
STEFKA KALOYANOVA等: ""Water-Soluble NIR-Absorbing Rylene Chromophores for Selective Staining of Cellular Organelles"", 《J.AM.CHEM.SOC.》 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112300213A (zh) * | 2020-10-29 | 2021-02-02 | 赤峰学院 | 固态/溶液中可逆变色可靶向线粒体的荧光染料及制备方法和应用 |
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