CN109180568A - 一种识别线粒体并具有双光子性质的荧光探针及应用 - Google Patents

一种识别线粒体并具有双光子性质的荧光探针及应用 Download PDF

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CN109180568A
CN109180568A CN201811072422.4A CN201811072422A CN109180568A CN 109180568 A CN109180568 A CN 109180568A CN 201811072422 A CN201811072422 A CN 201811072422A CN 109180568 A CN109180568 A CN 109180568A
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林伟英
王伟珊
刘勇
牛杰
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University of Jinan
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Abstract

本发明公开了一种在细胞内识别线粒体的双光子荧光探针FVPI，其化学结构式如式（I）所示。本发明还公开了所述荧光探针在检测过程中与商业线粒体染料相重合的性质。实验证明：本发明的荧光探针具有双光子成像性质且能够识别线粒体，该探针在活细胞靶标分子的标记领域具有潜在的应用价值。

Description

一种识别线粒体并具有双光子性质的荧光探针及应用
技术领域
本发明涉及一种识别线粒体并具有双光子性质的新型荧光探针及其应用，尤其涉及一种专一识别线粒体的芴-吡啶盐类化合物荧光探针及其应用；属于有机小分子荧光探针领域。
背景技术
线粒体由线粒体膜，线粒体膜间隙，线粒体内膜和线粒体基质这几个部分组成。线粒体参与各种细胞信号转导过程，并且是细胞能量的提供者。除了通过氧化磷酸化作用来产生能量外，线粒体在其他类型的新陈代谢中也起到至关重要的作用，包括血红素和铁硫合成，氨基酸和氮代谢，调节细胞中钩离子动态平衡等。此外，线粒体是细胞耗氧的主要区域及活性氧物种的主要来源。而活性氧物种与退行性疾病如阿尔兹海默症，帕金森症，缺血性和出血性中风神经元死亡，极性和慢性的退行性也脏细胞死亡及癌症有关。因此，为了解线粒体的生理学，监测细胞，组织和器官中线粒体化学物种是必要的。
近年来，荧光探针由于其高灵敏度，高选择性，实时检测及易操作性，被认为是用来检测生物制剂最强大的工具。双光子显微成像，通过在近红外波段内激发（约700-1100 nm）两个光子，提供了克服用短波长激发（约350-550 nm），光漂白，光损伤，及细胞自荧光的缺点。双光子探针具有能够在更低的探针浓度下检测，抑制对样品的光毒性损伤，减少自体荧光的干扰，同时能够更高分辨率进行活细胞成像的优点。因此，开发一个特定的，灵敏的，反应快的，可发出强烈的双光子激发荧光具有线粒体靶向的双光子荧光探针，是很有必要的。
发明内容
针对现有技术的不足，本发明要解决的问题是提供一种能识别线粒体的具有双光子性质的芴-吡啶盐类化合物荧光探针及其应用。
一种识别线粒体并具有双光子性质的荧光探针，所述荧光探针命名为FVPI，其化学结构式如式（I）所示：
（I）。
上述化合物的制备方法是：将2-溴芴与4-乙烯基吡啶在甲醇做溶剂条件下加热回流生成化合物3，然后将化合物3与碘乙烷以甲醇为溶剂加热回流得产物FVPI其NMR图谱见图1-2。
上述化合物的制备反应式如下：
上述荧光探针在检测线粒体变化的应用。
上述的应用中：所述荧光探针具有双光子成像的性质，分别在405nm及780nm处成像。
上述荧光探针与商用线粒体探针MTR、MTG相比，具有双光子成像的性质。同时在细胞中对其做了共定位实验。实现该探针对线粒体的识别作用。
实验证实：在细胞内进行成像时，当激发波长为405nm时，探针在细胞内有强烈的荧光；采用激发波长为780nm时，探针在细胞内仍有强烈的荧光探针（图6），说明探针具有能进行双光子成像的性质。因此探针大大降低了探针对人体的危害性，同时也消除了自身的干扰性且分辨率更高。另外通过共定位实验（图5）证实，通过与商用线粒体探针进行拟合，该探针能明显地定位于线粒体中，从而实现了线粒体内的检测。
本发明的有益效果
1、双光子成像
细胞内进行成像时，当激发波长为405nm、780nm时，探针在细胞内有强烈的荧光，即可实现双光子成像，大大降低了探针对人体的危害性，同时也消除了自身的干扰性且分辨率更高。
2、合成简单，检测性好
本发明所述的识别线粒体且具有能进行双光子成像的性质的芴-吡啶盐类化合物荧光探针是一类新型的高选择性识别线粒体的双光子荧光探针分子，该探针合成路径简便，易于应用，通过与商用线粒体探针进行拟合，该探针能明显地定位于线粒体中，共定位系数高达0.9，从而实现了线粒体内的检测。
3、为生物学成像应用奠定了理论基础
基于本发明提供的识别线粒体且具有双光子性质的芴-吡啶盐类化合物荧光探针能实现线粒体检测，并且实现细胞内成像，为生物学成像应用奠定了理论基础，预示其在荧光生物标记领域具有潜在的应用价值。且该化合物能够进行双光子成像，将为以后新型荧光探针的合成提供了新的发展方向。
附图说明
图1：化合物3 1H NMR (400 MHz, CDCl3)。
图2：化合物FVPI1H NMR(400 MHz, MeOD)。
图3：探针的溶剂化效应。
图4：探针的紫外吸收光谱。
图5：细胞的共定位实验。
图6：探针的双光子细胞生物成像应用。
具体实施方式
实施例1
化合物3的合成：
将2-溴芴1.0 g(4.07 mmol)，三（邻甲苯基）膦248.34mg(0.81mmol),醋酸钯 91.58mg(0.407mmol),加入两口烧瓶中，加入30mLTEA与15mL的DMF(v:v=1:1)溶解，氮气保护，室温搅拌约20分钟，加入4-乙烯基吡啶1.2g(12.21mmol), 95℃反应48h,冷却至室温，蒸出TEA，剩余物用二氯甲烷萃取，二氯甲院层依次用水，饱和食盐水洗涤，然后用无水MgSO4干燥，过滤，减压蒸出溶剂，粗产品用石油醚：乙酸乙酯（2:1）作淋洗液，经过柱色谱分离得到化合物3，产率约40%。
化合物FVPI的合成：
将0.30g(1.1mmol)化合物3，0.34g（2.2 mmol）碘乙烷，溶于15mL的乙醇中，在氮气保护下加热回流约20h，反应结束后冷却至室温，将溶液旋出。粗产品用DCM：MeOH（10:1）作淋洗液，经过柱色谱分离得到黄色产物。产率约65%。1H NMR (400 MHz, DMSO) δ8.99 (d, J =8.0Hz, 2H), 8.27 (d, J = 8.0Hz, 2H), 8.16 (d, J = 16.0Hz, 1H), 8.02 (m, 3H),7.80 (d, J=8.0Hz, 1H), 7.63 (t, J=12.0Hz, 2H), 7.42 (m, 2H), 4.55 (q, J =6.0Hz, 2H), 4.03 (s, 2H), 1.54 (t, J = 8.0Hz,3H).
实施例2
化合物FVPI的溶剂化效应
预先准备1份8 mL的10-3 M探针的N, N-二甲基甲酰胺溶液，然后分别取12μL加入六个相同的5mL容量瓶中，分别用DMF、EtOH、MeCN、PBS、MeOH、H2O稀释到3mL，然后进行荧光检测，结果见图3；由图3可知，探针在520nm左右有最大发射峰。
实施例3
化合物FVPI在不同溶剂下的紫外吸收
预先准备1份8 mL的10-3 M探针的N, N-二甲基甲酰胺溶液，然后分别取12μL加入六个相同的5mL容量瓶中，分别用DMF、EtOH、MeCN、PBS、MeOH、H2O稀释到3mL，然后进行紫外吸收检测，结果见图4；由图4可知，探针在390nm左右有最大吸收峰。
实施例4
生物成像：活细胞染色实验
将探针配成1 mM DMF母液，染色时用1mL培养基稀释。
将接种好的细胞在设定量浓度的探针分子溶液37ºC孵育30 min，用PBS洗3-5次，贴壁生长的细胞置于载玻片上；然后用荧光显微镜进行荧光成像(图5-B)。激发波长为405nm，吸收在420-470nm。
商用线粒体探针(MTR)进行共定位实验，由共聚焦荧光显微镜下成像图可知（图5-C），该试验证明了探针能很好的定位于线粒体（见图5）。共定位系数高达0.9，说明探针成功定位于线粒体中。
实施例5
双光子成像
将探针配成1 mM DMF母液，染色时用1mL培养基稀释。
将接种好的细胞在设定量浓度的探针分子溶液37ºC孵育30 min，用PBS洗3-5次，贴壁生长的细胞置于载玻片上；然后用荧光显微镜进行荧光成像。激发波长为780nm，吸收在420-470nm。

Claims (3)

1.一种识别线粒体并具有双光子性质的荧光探针，其特征在于，所述荧光探针命名为FVPI，其化学结构式如式（I）所示：
（I）。
2.如权利要求1所述的荧光探针的制备方法，其特征在于，方法为：将2-溴芴与4-乙烯基吡啶在甲醇做溶剂条件下加热回流生成化合物3，然后将化合物3与碘乙烷以甲醇为溶剂加热回流得产物FVPI，反应过程如下：
一种权利要求1所述的荧光探针在检测线粒体变化上的应用。
3.根据权利要求3所述的应用，其特征在于，所述荧光探针具有双光子成像的性质，分别在405nm及780nm处成像。
CN201811072422.4A 2018-09-14 2018-09-14 一种识别线粒体并具有双光子性质的荧光探针及应用 Pending CN109180568A (zh)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109810538A (zh) * 2019-02-02 2019-05-28 南京师范大学 一种线粒体定位染料及其制备方法和应用
CN112079771A (zh) * 2020-09-23 2020-12-15 华中科技大学 一种水溶性红色荧光的线粒体靶向探针及应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105062467A (zh) * 2015-08-07 2015-11-18 山东大学 一种转子型的双红线粒体荧光探针及其应用
CN107337640A (zh) * 2017-07-24 2017-11-10 广东工业大学 一种芴修饰8‑羟基喹啉类反应型荧光探针及其制备方法和应用
CN108210938A (zh) * 2017-12-26 2018-06-29 南京邮电大学 一种多功能靶向纳米荧光探针及其制备与应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105062467A (zh) * 2015-08-07 2015-11-18 山东大学 一种转子型的双红线粒体荧光探针及其应用
CN107337640A (zh) * 2017-07-24 2017-11-10 广东工业大学 一种芴修饰8‑羟基喹啉类反应型荧光探针及其制备方法和应用
CN108210938A (zh) * 2017-12-26 2018-06-29 南京邮电大学 一种多功能靶向纳米荧光探针及其制备与应用

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109810538A (zh) * 2019-02-02 2019-05-28 南京师范大学 一种线粒体定位染料及其制备方法和应用
CN112079771A (zh) * 2020-09-23 2020-12-15 华中科技大学 一种水溶性红色荧光的线粒体靶向探针及应用
CN112079771B (zh) * 2020-09-23 2022-03-18 华中科技大学 一种水溶性红色荧光的线粒体靶向探针及应用

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Application publication date: 20190111