CN106432334B - 具有双荧光发射的线粒体荧光探针及其制备方法和应用 - Google Patents
具有双荧光发射的线粒体荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一类线粒体荧光探针及其制备方法和应用,属于功能材料领域。所述线粒体荧光探针具有双重荧光发射功能,是一种季膦盐类化合物,其结构式如式I所示。式I中R为烷基或芳基,阴离子X为卤素离子。本发明中所述的荧光探针具有非常高效的线粒体特异性识别能力,荧光探针分子在不同光的激发下呈现出双重荧光发射现象,从而实现了线粒体精准的双重荧光标记。本发明中所述的荧光探针分子还具有优异的光稳定性,受线粒体表面电荷改变的影响较小,能够实现活细胞中线粒体分布和形态实时视踪。此外,本发明提供的系列线粒体荧光探针具有水溶性好、合成路线简捷、后处理简单、可以批量生产、成本较低等优势,表现出广阔的市场前景。
Description
技术领域
本发明属于功能材料领域,特别涉及一种具有双荧光发射的线粒体荧光探针及其制备方法和应用。
技术背景
细胞器在正常细胞的功能表达以及疾病发展过程中发挥着极其重要的作用。线粒体是一种重要真核细胞器,是细胞进行有氧呼吸的重要场所。线粒体除了给细胞供应能量外,其还参与了细胞信息传递、细胞分化、细胞凋亡以及调控细胞生长和细胞周期等生理过程。因此,发展一类对线粒体特异性荧光探针来视踪线粒体在细胞内的形态和分布,将有助于我们研究一些重要的细胞生理过程。
荧光成像技术因其具有高的空间和时间分辨性,目前已被广泛用于各种生理过程的监测,开发新型线粒体荧光探针也受到了高度关注。目前市售的线粒体荧光探针种类较少,价格较为昂贵,光稳定性还有待进一步提高。近年来,有一些新型线粒体荧光探针相继被报道,但所报道的线粒体荧光探针仍普遍存在结构复杂,合成困难等缺点。此外,具有双重荧光发射性能的线粒体荧光探针就可以对线粒体进行双重定位,减少背景荧光的干扰,从而可以使探针的选择性更高,定位更准确。目前市售及文献报道的线粒体荧光探针一般为单重荧光发射的分子探针,因而,急需开发一类具有双重荧光发射的线粒体荧光探针。
季膦盐结构是一类非常重要的线粒体靶向位点,通过对具有优异荧光性能的已有荧光团进行季膦盐结构的修饰,便可以高效、批量地制备一系列具有优异线粒体识别能力和优异荧光性能的线粒体荧光探针。
发明内容
本发明的目的在于提供一种具有双荧光发射的线粒体荧光探针及其制备方法和应用。本发明所述的线粒体荧光探针为季膦盐型分子探针,其分子结构包括苝基荧光骨架和具有线粒体识别性能的季膦盐基团,其中荧光骨架和季膦盐基团通过亚甲基基团相连。
本发明通过以下技术方案来实现:
一种具有双荧光发射的线粒体荧光探针,其中发挥主要功能的季膦盐化合物的分子结构如式I所示:
……………………………………(式I)
其中,其中R1,R2,R3独立为烷基或芳基;阴离子X为卤素离子。
作为优选,所述结构式中R1,R2,R3独立为C1-C8的烷基或苯基。进一步的优选为R1=R2=R3=丁基。
作为优选,所述结构式中阴离子X优选溴离子或氯离子,进一步优选溴离子。
本发明所述的具有双重荧光发射的线粒体荧光探针的最优选结构之一为三丁基(3-苝苄基)溴化磷。
本发明还提供了一种所述季膦盐型有机小分子压致变色材料的制备方法,通过苝苄基卤化物与三取代膦化合物反应制得,其制备反应式如下:
具体包括以下步骤:
a、将苝苄醇与二氯亚砜或三溴化磷于-10-80 ºC下反应0.5-24小时生成相应的苝苄基卤化物;
b、将所得的苝苄基卤化物与三取代膦化合物在溶剂中,于室温至120 ºC下反应2-24小时即可得到目标化合物;
作为优选,步骤a中,苝苄醇化合物与二氯亚砜或三溴化磷的摩尔比为1:1~1:4;所用反应溶剂为甲苯、四氢呋喃、二氯甲烷、氯仿、四氯化碳中的任意一种。
作为优选,步骤b中,苝苄基卤化物与三取代膦化合物的摩尔比为1:0.5~1:3;所用反应溶剂为甲醇、乙醇、丙酮、乙酸乙酯、N,N-二甲基甲酰胺、二甲亚砜、乙腈中的任意一种,优选乙酸乙酯。
本发明所述线粒体荧光探针的制备具有合成简捷,后处理简单等优点。
本发明还提供上述线粒体荧光探针对活细胞中的线粒体的标记及线粒体形态视踪的应用。所述活细胞为MG63细胞株、HUVEC细胞株、HepG2细胞株和Hela细胞株等。
本说明书中公开的所有特征,或公开的所有方法或过程中的步骤,除了互相排斥的特征和/或步骤以外,均可以以任何方式组合。
本发明的有益效果:
本发明中所述的荧光探针具有非常高效的线粒体特异性识别能力,荧光探针分子在不同光的激发下呈现出双重荧光发射现象,从而实现了线粒体精准的双重荧光标记。本发明中所述的荧光探针分子还具有优异的光稳定性,受线粒体表面电荷改变的影响较小,能够实现活细胞中线粒体分布和形态实时视踪。此外,本发明提供的系列线粒体荧光探针具有水溶性好、合成路线简捷、后处理简单、可以批量生产、成本较低等优势,表现出广阔的市场前景。
附图说明:
图1:三丁基(3-苝苄基)溴化磷的氢谱图。
图2:MG63细胞与三丁基(3-苝苄基)溴化磷(3.0 μM)共培养1小时的共聚焦荧光照片:(a)405 nm光激发蓝色荧光;(b)488 nm光激发绿色荧光;(c)明场照片。
图3:MG63细胞与三丁基(3-苝苄基)溴化磷(3.0 μM)和市售线粒体荧光探针Mito-Tracker Red FM共染的共聚焦荧光照片:(a)三丁基(3-苝苄基)溴化磷(3.0 μM,1 h),405nm光激发蓝色荧光;(b)三丁基(3-苝苄基)溴化磷(3.0 μM,1 h),488 nm光激发绿色荧光;(c)Mito-Tracker Red FM,552 nm光激发红色荧光;(d)图a和图c的叠加图荧光呈紫色;(e)图b和图c的叠加图荧光呈黄绿色;(f)发明场照片。
具体实施方式:
以下通过实施例的具体实施方式再对本发明的上述内容作进一步的详细说明。应当理解,此处所描述的具体实例仅仅用以解释本发明,并不用于限定本发明。在不脱离本发明的精神和原则之内做的任何修改,以及根据本领域普通技术知识和惯用手段做出的等同替换或者改进,均应包括在本发明的保护范围内。
实施例1:中间体苝苄溴的合成
于室温下,将三溴化磷(0.49 g)加入苝苄醇(0.42 g)的四氯化碳(50 mL)溶液中。升温至回流,反应2小时后停止反应。移除四氯化碳后,所得固体经甲醇(40 mL)洗涤、抽滤和真空干燥后得到橙色目标物苝苄溴(0.37 g,73%)。
实施例2:中间体苝苄氯的合成
于室温下,将二氯亚砜(0.4 mL)加入苝苄醇(0.3 g的甲苯(9 mL)溶液中。继续反应24小时后,停止反应。减压移除过量的二氯亚砜,残余物经水洗涤、抽滤和真空干燥后得目标物为浅黄色固体(0.31 g,97%)。
实施例3:目标物三丁基(3-苝苄基)溴化磷的合成
于氮气下,将苝苄溴(0.69 g)、三丁基膦(0.75 mL)和乙酸乙酯(15 mL)加入反应瓶中。升温至80 ºC下反应8小时后停止反应。抽滤,固体用乙酸乙酯洗涤得目标化合物三丁基(3-芘苄基)溴化磷为黄色固体(0.90 g,82%)。
实施例4:目标物三丁基(3-苝苄基)氯化磷的合成
在实施例3中将苝苄溴换成苝苄氯成功制备三丁基(3-苝苄基)氯化磷。
实施例5:
在实施例1-4中将苝苄醇换成芘苄醇分别制得三丁基(3-芘苄基)溴化磷和三丁基(3-芘苄基)氯化磷。
实施例6:目标物三丁基(3-苝苄基)溴化磷与MG63细胞的共培养
首先,将MG63细胞于37 ºC下培育24小时。随后,移除培养基,加入化合物三丁基(3-苝苄基)溴化磷(3 μM)的DMEM溶液,共培养1小时。用DMEM洗涤三次后,用荧光共聚焦显微镜观察得到图2。
图2a中的激发光为405纳米;图2b中的激发光为488纳米.从图2可以看出化合物三丁基(3-苝苄基)溴化磷能成功进入细胞并主要分布在细胞质中。此外,图2也清晰地表明,化合物三丁基(3-苝苄基)溴化磷在细胞内呈现出双重荧光发射现象。
实施例7:目标物三丁基(3-苝苄基)溴化磷与市售线粒体染料Mitotracker RedFM共定位分析
首先,将MG63细胞于37 ºC下培育24小时。随后,移除培养基,加入化合物三丁基(3-苝苄基)溴化磷(3 μM)的DMEM溶液,共培养1小时。用DMEM洗涤后,加入0.5 μM市售的线粒体染料Mitotracker Red FM,继续培养0.5小时。用DMEM洗涤三次后,用荧光共聚焦显微镜观察得到图3。
图3a中的激发光为405纳米;图3b中的激发光为488纳米;图3c中的激发光为522纳米。从叠加图3d和3e可以看出化合物三丁基(3-苝苄基)溴化磷的染色区域与市售线粒体染料Mitotracker Red FM的染色区域基本一致,说明化合物三丁基(3-苝苄基)溴化磷具有非常好的线粒体视踪能力。
实施例8:
在实施例1-7所述制备方法中,分别采用C1-C8的烷基膦或苯基膦代替三丁基膦。成功制得了一系列具有双重荧光发射的线粒体荧光探针。其中以实施例3中制备的产品综合性能最好。
实施例9:
在实施例1或4所述的制备方法中,调整相关参数进行系列实验:
在步骤a中分别选择-10℃、0℃、40℃、60℃、80℃代替室温反应,反应时间控制在0.5-24小时;分别控制苝苄醇化合物与三溴化磷的摩尔比为1:1、1:2和1:4;分别采用甲苯、二氯甲烷、氯仿、四氯化碳代替四氢呋喃。
在步骤b中分别选择20℃、50℃、120℃代替80℃反应,反应时间控制在2-24小时;分别控制苝苄基卤化合物与三取代膦化合物的摩尔比为1:0.5、1:1和1:3;分别采用甲醇、乙醇、丙酮、N,N-二甲基甲酰胺、二甲亚砜、乙腈代替乙酸乙酯。
通过实验参数筛选发现上述各条件下均可成功制备具有双重荧光发射的线粒体荧光探针,但仍以实施例3中所述的具体参数为最佳。
对比例1:
参照实施例6和7所述方法,分别采用实施例5制备三丁基(3-芘苄基)溴化磷和三丁基(3-芘苄基)氯化磷代替三丁基(3-苝苄基)溴化磷进行标记实验,结果显示实施例5中所得产品虽然也显示出一定的双荧光发射效果,但其发光强度等综合效果明显不如本发明中所述以苝苄基作为骨架的季膦盐型化合物。
以上所述仅为本发明的优选实施例,对本发明而言,仅仅是说明性的,而非限制性的;本领域普通技术人员理解,在本发明专利要求所限定的范围内,可对其进行许多改变、修改,甚至等效变更,但都将落入本发明的保护范围。
Claims (9)
1.一种具有双荧光发射的线粒体荧光探针,其特征在于,含有分子结构如式I所示的季膦盐类化合物:
其中R1,R2,R3独立为C1-C8的烷基或苯基;阴离子X为卤素离子。
2.根据权利要求1所述的具有双荧光发射的线粒体荧光探针,其特征在于,所述结构式中R1=R2=R3=丁基。
3.根据权利要求1所述的具有双荧光发射的线粒体荧光探针,其特征在于,所述结构式中阴离子X为溴离子或氯离子。
4.根据权利要求1所述的具有双荧光发射的线粒体荧光探针,其特征在于,所述季膦盐类化合物具体为三丁基(3-苝苄基)溴化磷。
5.一种权利要求1所述的具有双荧光发射的线粒体荧光探针的制备方法,其特征在于,具体包括以下步骤:
a、将苝苄醇与二氯亚砜或三溴化磷于-10-80℃下反应0.5-24小时生成相应的苝苄基卤化物;
b、将所得的苝苄基卤化物与三取代膦化合物在溶剂中,于室温至120℃下反应2-24小时即可得到目标化合物。
6.根据权利要求5所述的制备方法,其特征在于,步骤a中,苝苄醇化合物与二氯亚砜或三溴化磷的摩尔比为1:1~1:4;所用反应溶剂为甲苯、四氢呋喃、二氯甲烷、氯仿、四氯化碳中的任意一种。
7.根据权利要求5所述的制备方法,其特征在于,步骤b中,苝苄基卤化物与三取代膦化合物的摩尔比为1:0.5~1:3;所用反应溶剂为甲醇、乙醇、丙酮、乙酸乙酯、N,N-二甲基甲酰胺、二甲亚砜、乙腈中的任意一种。
8.一种权利要求1所述的具有双荧光发射的线粒体荧光探针的应用,其特征在于,将其用于对活细胞中的线粒体的标记及线粒体形态视踪。
9.根据权利要求8所述的应用,其特征在于,所述活细胞为MG63细胞株、HUVEC细胞株、HepG2细胞株和Hela细胞株中的至少一种。
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