CN112047979A - 荧光探针Mito-HNO及其制备方法与其在检测线粒体中HNO的应用 - Google Patents
荧光探针Mito-HNO及其制备方法与其在检测线粒体中HNO的应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及线粒体靶向定位检测技术领域,具体涉及荧光探针Mito-HNO及其制备方法与其在检测线粒体中HNO的应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
线粒体广泛存在于大多数细胞当中,是细胞制造能量的亚细胞器结构,是有氧呼吸的主要场所。通过氧化呼吸链提供能量从而在许多重要的细胞过程中发挥着作用。例如ATP的产生、中间代谢、钙调控和氧化还原信号传导以及细胞的凋亡过程。线粒体是细胞中对各种氧化损伤最为敏感的细胞器,当细胞受到损伤时,线粒体的大小、数量以及结构都会发生变化。因此,当疾病发生时,及时的监测线粒体内活性分子的变化情况有助于许多疾病的早期诊断。
亚硝酰氢(HNO)作为一种NO的类似物,在某些情况下能够直接通过一氧化氮合成酶产生,并且HNO与NO能够在超氧化物歧化酶(SOD)存在下相互转化。HNO在细胞内发挥着重要的作用,例如它能够与蛋白质巯基反应抑制醛脱氢酶的活性、激活哺乳动物血管系统中电压依赖性的K+通道以及在治疗心血管疾病中担任重要角色等等。
但由于HNO是一种能够自发反应的物质,在生物体内存在时间短极难捕获,因此直接检测HNO的方法及手段仍需进一步发展,这也限制了活细胞及体内环境许多生理病理过程中HNO作用研究的进行。开发用于检测生物体内HNO的高灵敏度、高选择性的工具非常必要,有助于推进HNO在生物体内相关信号传导及通路的研究。
靶向线粒体检测线粒体内的HNO有助于疾病的早期诊断,为临床病理检测提供可靠数据,因此发展成像线粒体内HNO的荧光探针是具有极大指导意义的。
发明内容
为了解决现有技术中存在的技术问题,本发明的目的是提供一种荧光探针Mito-HNO及其制备方法与其在检测线粒体中HNO的应用,荧光探针Mito-HNO能够靶向定位线粒体,且其发射光位于近红外区,具有组织穿透深度深,利于活体成像等诸多优势,生物相容性好,对细胞和活体损伤小。
具体地,本发明的技术方案如下所述:
在本发明的第一方面,提供一种荧光探针Mito-HNO,其结构式为:
在本发明的第二方面,提供第一方面所述荧光探针Mito-HNO的制备方法,以环己酮、2,3,3-三甲基假吲哚、间苯二酚、2-苯基磷苯甲酸为原料,按照如下反应路线进行反应制备:
在本发明的第三方面,提供第一方面所述荧光探针Mito-HNO在靶向定位线粒体检测HNO中的应用。
在本发明的第四方面,提供一种线粒体中HNO的检测方法,具体的,将培养好的细胞放置于含有第一方面所述的荧光探针Mito-HNO分子的缓冲溶液中孵育,孵育一定的时间后除去孵育液,将孵育后的细胞进行紫外吸收检测、荧光发射检测或共聚焦成像检测。
本发明的具体实施方式具有以下有益效果:
提供了一种线粒体靶向检测HNO的近红外荧光探针,该合成设计策略是具有普适性的,具体线粒体的靶向定位效果;
靶向定位线粒体检测HNO的近红外荧光探针Mito-HNO的发射光位于近红外区,具有组织穿透深度深,利于活体成像等诸多优势;这使得其能够成功应用于动物模型中,是一种新型的成像工具,今后有望开发成为活体线粒体相关疾病中HNO检测的有力工具;
探针Mito-HNO的生物相容性好,对细胞和活体损伤小;
探针Mito-HNO具有光声性能,也可开发成为光声探针;
设计策略和合成路线简便,且原料均为廉价易得的,有望应用于市场化的生产。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1是本发明实施例1制备的荧光探针Mito-HNO与HNO反应前后的吸收光谱图,其中,横坐标为波长(nm),纵坐标为紫外吸收强度,黑色表示Mito-HNO,红色表示Mito-HNO+HNO;
图2是本发明实施例1制备的荧光探针Mito-HNO与细胞内常见的氨基酸、金属离子、活性氧及活性氮等成分的选择性测定柱状图;
图3是本发明实施例1制备的荧光探针Mito-HNO在人宫颈癌细胞(Hela细胞)中与四种市售的不同亚细胞器的商业化染料共染后的细胞荧光成像图;
图4是本发明实施例1制备的荧光探针在人宫颈癌细胞(Hela细胞)中的不同刺激效果下,细胞内HNO浓度的共聚焦荧光成像图;
图5是本发明实施例1制备的荧光探针在博来霉素诱导的急性肝损伤模型小鼠中的成像图。
具体实施方式
应该指出,以下详细说明都是例示性的,旨在对本发明提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本申请的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
正如背景技术中论述的,靶向线粒体检测线粒体内的HNO有助于疾病的早期诊断,为临床病理检测提供可靠数据,发展成像线粒体内HNO的荧光探针非常必要。鉴于此,本发明公开了荧光探针Mito-HNO及其制备方法与其在检测线粒体中HNO的应用。
本发明的一种实施方式中,提供一种荧光探针Mito-HNO,其结构式为:
本发明的一种实施方式中,提供一种上述荧光探针Mito-HNO的制备方法,以环己酮、2,3,3-三甲基假吲哚、间苯二酚、2-苯基磷苯甲酸为原料,按照如下反应路线进行反应制备:
在一种优选的实施方式中,羟基部花菁(化合物4)和2-二苯基膦苯甲酸反应的脱水缩合剂为二环己基碳二亚胺(DCC)及4-二甲氨基吡啶(DMAP);
4-二甲氨基吡啶(DMAP)为反应提供碱性反应条件的同时,还能够催化2-二苯基磷苯甲酸与二环己基碳二亚胺(DCC)形成活化羧基的中间体;
优选的,2-二苯基磷苯甲酸与二环己基碳二亚胺(DCC)形成活化羧基的中间体的温度为0-5℃;
优选的,4-二甲氨基吡啶(DMAP)的浓度为0.25-0.3mmol,二环己基碳二亚胺(DCC)的浓度为5-6mmol。
在一种优选的实施方式中,羟基部花菁(化合物4)和2-二苯基膦苯甲酸反应体系的溶剂为二氯甲烷,进一步优选的,二氯甲烷进行除水处理。
在一种优选的实施方式中,羟基部花菁(化合物4)的浓度为1-1.5mmol,2-二苯基磷苯甲酸的浓度为5-7.5mmol。
在一种优选的实施方式中,2-二苯基磷苯甲酸经活化后加入羟基部花菁(化合物4)后反应的温度为20-25℃,反应的时间为10-14h。
为了获得产率更高的探针Mito-HNO,选择增加2-二苯基磷苯甲酸形成的羧基中间体的浓度以提高产率。该步反应,传统的合成工艺中羟基化合物与羧基化合物多为1:1进行投料,但实验中我们发现,当2-二苯基磷苯甲酸的浓度增加到5倍时,产率明显提高。因此,该步的投料比改进为优选条件。
本发明的一种实施方式中,提供上述荧光探针Mito-HNO在靶向定位线粒体检测HNO中的应用。
荧光探针Mito-HNO遇到HNO时,探针结构中的酯基会通过亲核进攻并水解成为羟基;羟基对于荧光团部花菁具有ICT效应,使得给电子能力增强,荧光旨在724nm处发射增加,由于线粒体外膜带有正电荷,探针Mito-HNO带有正电荷,通过静电作用探针可以进入线粒体并对线粒体中的HNO进行检测。
为了使得本领域技术人员能够更加清楚地了解本发明技术方案,一下将结合具体的实施例详细说明本发明的技术方案。
实施例1
荧光探针的合成
羟基部花菁(物质4)的合成是根据已有报道合成的。取原料2-二苯基磷苯甲酸(5mmol)及二环己基碳二亚胺(5mmol)、4-二甲氨基吡啶(0.25mmol)溶于15mL二氯甲烷中,0℃下活化羧基30min,然后加入羟基部花菁(1mmol),室温下搅拌12h。反应完毕后,旋转蒸发除去溶剂。随后用二氯甲烷:甲醇=20:1作为洗脱剂,在柱层析法提纯得到蓝紫色固体Mito-HNO(57%)。
核磁及质谱表征:
1H NMR(400MHz,CDCl3)δ=8.71(d,J=15.2Hz,1H),8.36–8.31(m,1H),8.20(d,J=8.4Hz,1H),8.05(dd,J=18.2,8.5Hz,2H),7.73–7.66(m,2H),7.62–7.52(m,3H),7.39–7.24(m,12H),7.11(d,J=1.8Hz,1H),7.07–7.05(m,1H),6.83–6.76(m,2H),4.86(q,J=7.3Hz,2H),2.82(dt,J=63.9,5.7Hz,4H),2.07(s,6H),2.02–1.95(m,2H),1.62(t,J=7.2Hz,3H).13C NMR(101MHz,CDCl3)δ=179.08,159.49,152.41,145.32,138.42,136.56,134.73,134.13,133.92,133.16,133.10,131.65,131.57,130.49,130.41,130.34,128.97,128.71,128.64,128.60,128.21,127.97,127.66,126.54,122.32,119.79,119.04,115.54,111.89,109.63,106.01,77.37,77.26,77.05,76.73,52.67,42.99,29.71,29.53,27.82,25.09,20.19,14.13,13.67.HRMS(ESI)m/z:[M+]calculated for C50H43NO3P+,736.2981found736.3063.
效果实验:
通常,可以将染料分子溶解在生理盐水、缓冲液或由乙腈、二甲亚砜等水溶性有机溶剂,然后加入适当缓冲液及其他有机试剂进行测试。分别研究了探针Mito-HNO在2.5X且pH=7.4的磷酸缓冲水溶液及各种常见的有机试剂中的光物理性质并将其用于活细胞成像实验。活细胞的染色方法是将培养好的细胞放于含有探针分子的缓冲溶液中孵育,孵育一定的时间后除去孵育液,进行共聚焦成像实验。
探针Mito-HNO与HNO反应的紫外吸收、荧光发射及选择性实验:
对照组:Mito-HNO(10μM)、PBS缓冲溶液(25mM)、pH=7.4;实验组:Mito-HNO(10μM)、PBS缓冲溶液(25mM)、pH=7.4、HNO(50μM)。将对照组和实验组都在37℃下孵育20min,测量其紫外吸收光谱图,其光谱图显示于图1。横坐标为波长(nm),纵坐标为紫外吸收强度。图二为Mito-HNO对多种的生物学相关成分的响应情况,已检测的生物学相关成分包括生物硫醇(半胱氨酸、高半胱氨酸、谷胱甘肽)、盐(KCl、NaCl、CaCl2、FeCl3、MgCl2、ZnCl2)、活性氧、活性氮及自由基(NO、O2 ·-、ClO-、H2O2)和HNO。如图2所示,只有当HNO存在时,荧光强度有显著的增强且响应倍数高达6倍。这个说明与生物体内其他组分相比,Mito-HNO对HNO有极好的选择性,可以用在复杂的细胞及活体生物环境中,特异性检测HNO。
Mito-HNO的线粒体靶向性实验:
人宫颈癌细胞(Hela细胞)是由高糖的DMEM培养液培养的,分别加入2μM的探针以及0.5μM不同亚细胞器的商业化染料(包括高尔基体、线粒体、溶酶体、内质网四种)共孵育细胞30min后,使用激光共聚焦显微镜进行共定位成像实验。共定位细胞成像实验如图3所示,探针展现了优异的线粒体定位效果。
探针对活细胞共聚焦荧光成像实验:
人宫颈癌细胞(Hela细胞)是由高糖的DMEM培养液培养的,提前用各种条件处理(包括外加100μM HNO组以及外加500μM NaHS组),然后分别加入2μM的探针在37℃中孵育30分钟,然后将细胞带有探针孵育液洗掉,进行激光共聚焦荧光成像,如图4所示。外加HNO组,细胞中的HNO浓度上升,荧光强度更亮。外加NaHS组,由于NaHS是细胞内合成HNO的重要原料,与对照组相比HNO浓度明显上升,荧光强度更亮。图4中单光子激发光为633nm,荧光通道收集700-800nm。
探针对急性肝损伤小鼠活体成像实验:
实验组小鼠静脉注射博来霉素(37.5mg mL-1,100μL),对照组小鼠静脉注射等量生理盐水。28天后,在小鼠腹腔注射水合氯醛将其麻醉进行后续实验。静脉注射探针Mito-HNO(10μM)后取出小鼠的肝脏及其他器官进行荧光成像,如图5所示。静脉注射博来霉素的实验组小鼠的肝脏部位荧光明显比对照组荧光增强,说明探针Mito-HNO成功地应用于了急性肝损伤模型小鼠的活体成像中。
实施例2
荧光探针的合成
羟基部花菁(物质4)的合成是根据已有报道合成的。取原料2-二苯基磷苯甲酸(1mmol)及二环己基碳二亚胺(1mmol)、4-二甲氨基吡啶(0.05mmol)溶于15mL二氯甲烷中,0℃下活化羧基30min,然后加入羟基部花菁(1mmol),室温下搅拌12h。反应完毕后,旋转蒸发除去溶剂。随后用二氯甲烷:甲醇=20:1作为洗脱剂,在柱层析法提纯得到蓝紫色固体Mito-HNO(15%)。
实施例3
荧光探针的合成
羟基部花菁(物质4)的合成是根据已有报道合成的。取原料2-二苯基磷苯甲酸(3mmol)及二环己基碳二亚胺(3mmol)、4-二甲氨基吡啶(0.15mmol)溶于15mL二氯甲烷中,0℃下活化羧基30min,然后加入羟基部花菁(1mmol),室温下搅拌12h。反应完毕后,旋转蒸发除去溶剂。随后用二氯甲烷:甲醇=20:1作为洗脱剂,在柱层析法提纯得到蓝紫色固体Mito-HNO(24%)。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
3.根据权利要求2所述的荧光探针Mito-HNO的制备方法,其特征在于:羟基部花菁和2-二苯基膦苯甲酸反应的脱水缩合剂为二环己基碳二亚胺(DCC)及4-二甲氨基吡啶(DMAP)。
4.根据权利要求2所述的荧光探针Mito-HNO的制备方法,其特征在于:反应体系的溶剂为二氯甲烷;优选的,二氯甲烷进行除水处理。
5.根据权利要求2所述的荧光探针Mito-HNO的制备方法,其特征在于:羟基部花菁和2-二苯基磷苯甲酸的反应的温度为20-25℃,反应的时间为10-14h。
6.根据权利要求2所述的荧光探针Mito-HNO的制备方法,其特征在于:2-二苯基磷苯甲酸与二环己基碳二亚胺(DCC)形成活化羧基的中间体的温度为0-5℃,反应的时间为0.5h。
7.根据权利要求2所述的荧光探针Mito-HNO的制备方法,其特征在于:4-二甲氨基吡啶(DMAP)的浓度为0.25-0.3mmol,二环己基碳二亚胺(DCC)的浓度为5-6mmol。
8.根据权利要求2所述的荧光探针Mito-HNO的制备方法,其特征在于:羟基部花菁的浓度为1-1.5mmol,2-二苯基磷苯甲酸的浓度为5-7.5mmol。
9.权利要求1所述的荧光探针Mito-HNO在靶向定位线粒体检测HNO中的应用。
10.一种线粒体中HNO的检测方法,其特征在于,将培养好的细胞放置于含有权利要求1所述的荧光探针Mito-HNO分子的缓冲溶液中孵育,孵育一定的时间后除去孵育液,将孵育后的细胞进行紫外吸收检测、荧光发射检测或共聚焦成像检测。
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