CN112940037A - 一种用于线粒体靶向的双光子荧光探针及其合成方法与成像应用 - Google Patents
一种用于线粒体靶向的双光子荧光探针及其合成方法与成像应用 Download PDFInfo
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Abstract
本发明提供了一种用于线粒体靶向的双光子荧光探针及其合成方法与其在活细胞的双光子荧光成像中的应用。该荧光探针以萘酰亚胺为探针母体，4位引入能够强烈抑制分子内扭转的氮杂环酮类取代基，N位引入三苯基膦定位基团，能够实现对线粒体快速、准确标记(如下图式1所示)。此外，该类探针能够实现双光子荧光成像，在实时监测线粒体动态、了解线粒体的生物学和病理学等领域有很好的应用前景。
Description
技术领域
本发明属于荧光探针技术领域，具体涉及一种用于线粒体靶向的双光子荧光探针及其合成方法与成像应用。
背景技术
线粒体能够执行许多功能并负责氧化磷酸化的蛋白质，因此，线粒体一直被认为是研究人类生理疾病的主要细胞器。例如：线粒体功能的衰退与衰老、癌症和神经退行性疾病(包括阿尔茨海默氏症、帕金森氏病)直接相关；线粒体运输在能量供应方面也扮演着重要角色。因此，线粒体的动态、实时监测，对于了解线粒体的生物学和病理学作用十分重要。
小分子荧光探针由于其空间体积较小，标记速度快，颜色丰富等特点，已逐渐成为分子成像研究中的强大工具。双光子显微镜使用两个近红外的低能量光子作为激发光，具有以下优点：(1)更深的组织穿透力(>500μm)，自吸收小，自体荧光的干扰小；(2)双光子激发本质上是局部的，因此可以提供更高的空间分辨率；(3)双光子激发由于能量较少，因此可以使探针的光漂白和生物样品的光损伤最小化。虽然小分子荧光探针、双光子荧光成像有诸多优点，然而双光子荧光探针种类仍然匮乏，这也极大地限制了双光子荧光成像技术在线粒体相关研究领域的深入应用。因此，开发高性能的双光子线粒体荧光探针对于活细胞内线粒体的动态监测、功能行使等方面的研究显得尤为迫切。
发明内容
本发明的目的是提供一类用于活细胞线粒体靶向荧光探针的合成及基于细胞的双光子荧光成像研究。
本发明提供了一类新型双光子线粒体标记的荧光探针，该荧光探针以萘酰亚胺为探针母体，在其4位引入能够强烈抑制分子内扭转的氮杂环酮类取代基，N位引入三苯基膦定位基团，借助线粒体自身的膜电位实现对线粒体快速、准确的定位，从而进行双光子荧光成像研究。
其中，三苯基膦定位基团结构式为
一种用于线粒体靶向的双光子荧光探针，结构如下：
其中R为氮杂环酮类取代基，氮杂环包括四元氮杂环、五元氮杂环、六元氮杂环等。
一种用于线粒体靶向的双光子荧光探针的合成方法，其合成路线，如下：
具体合成步骤如下：
(1)中间体Mito-Br的合成
将4-溴-1,8-萘酐溶于乙醇中，而后向该反应液中加6-三苯基膦-1-己胺溴盐。将反应液加热至55-115℃，搅拌8-14h。冷却降温后，减压除去溶剂，残余物经硅胶柱分离(展开剂为石油醚：甲醇＝50:1-20:1)得白色固体Mito-Br。
(2)探针Mito-405的合成
Mito-Br、氮杂环酮RH、Cs2CO3、G3-Xantphos(XantPhos Pd G3 95％)置于双口瓶中并用氮气置换3-4次。向反应体系中加入干燥的二氧六环，并加热至70-130℃。13-20h后减压除去溶剂，残余物经硅胶柱分离得固体(荧光探针)。
步骤(1)中，4-溴-1,8-萘酐：6-三苯基膦-1-己胺溴盐
的质量比为1-3:5；4-溴-1,8-萘酐的质量与乙醇的体积比为1:36-110g/mL。
所述氮杂环酮RH为
或
步骤(2)中，中间体Mito-Br：氮杂环酮(RH)的质量比为1:0.5-1.5；Cs2CO3与氮杂环酮(RH)的摩尔比为1:1；G3-Xantphos(XantPhos Pd G3 95％)为Mito-Br的1mol％；中间体Mito-Br的质量与二氧六环的体积比为1:80-160g/mL。
一类新型线粒体探针能够对线粒体进行快速、准确地特异性识别。
一类新型线粒体靶向的荧光探针在细胞、组织样本中进行研究。
一类新型线粒体靶向的荧光探针在双光子荧光成像中的应用。
本发明优点：
该类探针原料廉价易得，合成方法简单、产物易分离等优点。
该类探针的荧光激发波长、发射波长长，吸光度大，能够在低浓度下，对线粒体快速、准确地特异性标记并进行双光子荧光成像。该类探针促进了双光子荧光成像技术在线粒体相关研究领域的应用。
附图说明
图1为实施例1制备的Mito-405的核磁共振氢谱。
图2为实施例1制备的线粒体探针Mito-405在水中的激发光谱图与荧光发射谱图，横坐标为波长，纵坐标为归一化荧光强度，荧光探针的浓度为10μM。
图3为实施例1制备的探针Mito-405在Hela细胞中的单光子荧光成像图，荧光探针的浓度为1μM。
图4为实施例1制备的探针Mito-405在Hela细胞中的双光子荧光成像图，荧光探针的浓度为1μM。
具体实施方式
本发明提供了一类线粒体靶向的荧光探针及其制备方法和在荧光方面的应用。
下面将对本发明实施例中的方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
下面三个实施(实施例1-3)例为Mito-405的制备，其反应式如下所示：
实施例1.
线粒体探针Mito-405的合成方法。
(1)中间体Mito-Br的合成
将4-溴-1,8-萘酐(277.07mg,1.00mmol)溶于9.97mL乙醇中，而后向该反应液中加6-三苯基膦-1-己胺溴盐
(1385.35mg，3.13mmol)。将反应液加热至55℃，搅拌8h。冷却降温后，减压除去溶剂，残余物经硅胶柱分离(展开剂为石油醚：甲醇＝50:1-20:1)得白色固体Mito-Br 462.94mg，产率66％。
(2)探针Mito-405的合成
Mito-Br(84.17mg，0.12mmol)，2-氮杂环丁酮(42.09mg，0.59mmol)，Cs2CO3(192.23mg,0.59mmol),G3-Xantphos(XantPhos Pd G3 95％)(1mol％)置于双口瓶中并用氮气置换3-4次。向反应体系中加入7mL干燥的二氧六环，并加热至70℃。13h后减压除去溶剂，残余物经硅胶柱分离(展开剂为二氯甲烷：甲醇＝30:1-10:1)得白色固体48.13mg，产率58％。
其核磁谱图氢谱如图1所示，具体数据如下：
1H NMR(400MHz,CDCl3)δ8.82(dd,J＝8.6,0.9Hz,1H),8.59(d,J＝7.2Hz,1H),8.53(d,J＝8.1Hz,1H),7.92–7.64(m,16H),7.62(d,J＝8.1Hz,1H),4.09(dd,J＝11.0,6.1Hz,4H),3.85–3.70(m,2H),3.32(t,J＝4.8Hz,2H),1.76–1.61(m,8H).
经检测，其结构如上式Mito-405所示，能够快速、准确定位于活细胞线粒体，满足双光子荧光成像的需求，其性能如下：
将该探针Mito-405溶解于DMSO溶液中，配制成浓度为2mM的Mito-405母液，取20μL母液，加入4mL去离子水中，配制成10μM的荧光探针测试液，并进行荧光光谱的测试。Mito-405在水中的荧光激发与发射归一化谱图如图2所示：在水中的荧光激发波长为374nm，荧光发射波长为476nm，且荧光具有很好的稳定性。
实施例2.
线粒体探针Mito-405的合成方法。
(1)中间体Mito-Br的合成
将4-溴-1,8-萘酐(277mg,1.00mmol)溶于20.23mL乙醇中，而后向该反应液中加6-三苯基膦-1-己胺溴盐
(692.50mg，1.57mmol)。将反应液加热至85℃，搅拌11h。冷却降温后，减压除去溶剂，残余物经硅胶柱分离(展开剂为石油醚：甲醇＝50:1-20:1)得白色固体Mito-Br 505.03mg，产率72％。
(2)探针Mito-405的合成
Mito-Br(84.17mg，0.12mmol)，2-氮杂环丁酮(84.17mg，1.18mmol)，Cs2CO3(384.47mg，1.18mmol),G3-Xantphos(XantPhos Pd G3 95％)(1mol％)置于双口瓶中并用氮气置换3-4次。向反应体系中加入10.10mL干燥的二氧六环，并加热至100℃。16.5h后减压除去溶剂，残余物经硅胶柱分离(展开剂为二氯甲烷：甲醇＝30:1-10:1)得白色固体51.45mg，产率62％。
经检测，其结构如上式Mito-405所示。它的荧光性能如下：
荧光探针Mito-405的浓度为10μM，在去离子水中的荧光激发波长为374nm，荧光发射波长为476nm，且荧光具有很好的稳定性。
实施例3.
线粒体探针Mito-405的合成方法。
(1)中间体Mito-Br的合成
将4-溴-1,8-萘酐(277.07mg,1.00mmol)溶于30.48mL乙醇中，而后向该反应液中加6-三苯基膦-1-己胺溴盐
(461.78mg，1.04mmol)。将反应液加热至115℃，搅拌14h。冷却降温后，减压除去溶剂，残余物经硅胶柱分离(展开剂为石油醚：甲醇＝50:1-20:1)得白色固体Mito-Br 547.12mg，产率78％。
(2)探针Mito-405的合成
Mito-Br(84.17mg，0.12mmol)，2-氮杂环丁酮(126.26mg，1.78mmol)，Cs2CO3(579.96mg，1.78mmol),G3-Xantphos(XantPhos Pd G3 95％)(1mol％)置于双口瓶中并用氮气置换3-4次。向反应体系中加入13.47mL干燥的二氧六环，并加热至130℃。20h后减压除去溶剂，残余物经硅胶柱分离(展开剂为二氯甲烷：甲醇＝30:1-10:1)得白色固体57.26mg，产率69％。
经检测，其结构如上式Mito-405所示。它的荧光性能如下：
荧光探针Mito-405的浓度为10μM，在去离子水中的荧光激发波长为374nm，荧光发射波长为476nm，且荧光具有很好的稳定性。
实施例4
本实施例进行荧光探针Mito-52的制备，其反应式如下所示：
(1)中间体Mito-Br的合成
将4-溴-1,8-萘酐(277mg,1.00mmol)溶于20.23mL乙醇中，而后向该反应液中加6-三苯基膦-1-己胺溴盐
(692.50mg，1.57mmol)。将反应液加热至85℃,搅拌11h。冷却降温后，减压除去溶剂，残余物经硅胶柱分离(展开剂为石油醚：甲醇＝50:1-20:1)得白色固体Mito-Br 505.03mg，产率72％。
(2)探针Mito-52的合成
Mito-Br(84.17mg,0.12mmol)，2-吡咯烷酮(84.17mg,0.99mmol)，Cs2CO3(322.56mg,0.99mmol)，G3-Xantphos(XantPhos Pd G3 95％)(1mol％)置于双口瓶中并用氮气置换3-4次。向反应体系中加入10.17mL干燥的二氧六环，并加热至100℃。16.5h后减压除去溶剂，残余物经硅胶柱分离得固体54.19mg，产率64％。
Mito-52的质谱具体数据如下：
C40H38BrN2O3P[M+H]+理论值：705.1805，实际值：705.1832。
经检测，其结构如Mito-52所示。
实施例5
本实施例进行荧光探针Mito-62的制备，其反应式如下所示：
(1)中间体Mito-Br的合成
将4-溴-1,8-萘酐(277mg,1.00mmol)溶于20.23mL乙醇中，而后向该反应液中加6-三苯基膦-1-己胺溴盐
(692.50mg，1.57mmol)。将反应液加热至85℃，搅拌11h。冷却降温后，减压除去溶剂，残余物经硅胶柱分离(展开剂为石油醚：甲醇＝50:1-20:1)得白色固体Mito-Br 505.03mg，产率72％。
(2)探针Mito-62的合成
Mito-Br(84.17mg，0.12mmol)，2-氮己环酮(84.17mg，0.85mmol)，Cs2CO3(276.95mg，0.85mmol),G3-Xantphos(XantPhos Pd G3 95％)(1mol％)置于双口瓶中并用氮气置换3-4次。向反应体系中加入10.10mL干燥的二氧六环，并加热至100℃。16.5h后减压除去溶剂，残余物经硅胶柱分离得固体56.13mg，产率65％。
Mito-62的质谱具体数据如下：
C41H40BrN2O3P[M+H]+理论值：719.2067，实际值：719.2038。
经检测，其结构如Mito-62所示。
实施例6
Mito-405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分钟后分别对活细胞线粒体进行单光子荧光成像实验、双光子荧光成像实验。
Mito-405终浓度为1μM的单光子荧光成像实验如图3所示。Mito-405终浓度为1μM的双光子荧光成像实验如图3所示。从图中我们可以看到双光子荧光成像比单光子荧光成像更清晰，说明以Mito-405为代表的这类荧光探针荧光性能优异，可对活细胞内的线粒体进行特异性标记。
Claims (9)
1.一类用于线粒体靶向的双光子荧光探针，其特征在于，结构式如下所示，
其中，R为氮杂环酮类取代基，氮杂环包括四元氮杂环、五元氮杂环或六元氮杂环。
2.根据权利要求1所述的用于线粒体靶向的双光子荧光探针，其特征在于，该荧光探针以萘酰亚胺为探针母体，4位引入能够强烈抑制分子内扭转的氮杂环酮类取代基，N位引入三苯基膦定位基团。
3.根据权利要求2所述的用于线粒体靶向的双光子荧光探针，其特征在于，三苯基膦定位基团为
4.根据权利要求1所述的用于线粒体靶向的双光子荧光探针，其特征在于，该类探针结构式(1)为下列中的一种，
5.权利要求1-4任一所述用于线粒体靶向的双光子荧光探针的合成方法，其特征在于，该方法包含以下合成步骤：
(1)中间体Mito-Br的合成
将4-溴-1,8-萘酐溶于乙醇中，而后向该反应液中加6-三苯基膦-1-己胺溴盐
将反应液加热至55-115℃，搅拌8-14h；冷却降温后，减压除去溶剂，残余物经硅胶柱分离(展开剂为石油醚：甲醇＝50:1-20:1)得白色固体Mito-Br；
(2)荧光探针的合成
Mito-Br、氮杂环酮RH、Cs2CO3、G3-Xantphos(XantPhos Pd G3 95％)置于双口瓶中并用氮气置换3-4次；向反应体系中加入干燥的二氧六环，并加热至70-130℃；13-20h后减压除去溶剂，残余物经硅胶柱分离得固体(荧光探针)。
6.根据权利要求5所述的用于线粒体靶向的双光子荧光探针的合成方法，其特征在于：氮杂环酮RH为
7.根据权利要求5所述的用于线粒体靶向的双光子荧光探针的合成方法，其特征在于：步骤(1)中，4-溴-1,8-萘酐：6-三苯基膦-1-己胺溴盐
的质量比为1-3:5；4-溴-1,8-萘酐的质量与乙醇的体积比为1:36-110g/mL。
8.根据权利要求5所述的用于线粒体靶向的双光子荧光探针的合成方法，其特征在于步骤(2)中，中间体Mito-Br：氮杂环酮(RH)的质量比为1:0.5-1.5；Cs2CO3与氮杂环酮(RH)的摩尔比为1:1；G3-Xantphos(XantPhos Pd G3 95％)为Mito-Br的1mol％；
中间体Mito-Br的质量与二氧六环的体积比为1:80-160g/mL。
9.一种如权利要求1所述的用于线粒体靶向的双光子荧光探针在活细胞内对线粒体的双光子荧光成像领域的应用。
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