CN112062708B - 一种检测半胱氨酸的荧光探针化合物及其应用 - Google Patents
一种检测半胱氨酸的荧光探针化合物及其应用 Download PDFInfo
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Abstract
本发明涉及一种检测半胱氨酸的荧光探针化合物及其应用。该化合物如式Ⅰ所示。本发明以所述化合物作为检测Cys的荧光探针，提供了一种可用于环境、细胞和活体内选择性检测Cys的荧光探针,在Cys存在下荧光探针的荧光强度发生明显升高,可用于Cys的检测，并可大大降低外部检测条件的干扰，提高检测速度和精度。探针能被用来深入研究Cys在环境和生物体内的产生、转运及累积等过程的动力学机理，还能有助于阐明半胱氨酸在生理病理过程中的生物作用，研究半胱氨酸动态平衡的新机制。
Description
技术领域
本发明涉及一种检测半胱氨酸的荧光探针化合物及其应用。
背景技术
作为一种重要的生物巯基，半胱氨酸(cysteine,Cys)在生物有机体的多种生理过程中起着重要作用。正常Cys浓度维持在微摩尔水平，异常水平的细胞内半胱氨酸可导致许多疾病。Cys浓度异常增高可导致心血管疾病、神经毒性、阿尔茨海默病和帕金森等疾病，而其缺陷则会引起水肿、脱发和肝损伤等。细胞内大多Cys参与谷胱甘肽(GSH)等生物硫醇和其他生物分子的合成与代谢。此外，由于Cys与GSH和同型半胱氨酸(Hcy)有类似的化学结构和性质，Cys的检测总是受到GSH和Hcy的干扰。详尽的了解Cys在正常和疾病状态下的产生、分布和生理功能对阐明细胞信号转导机制有着重要的意义。
由于生物体内环境的多样性和复杂性，因此开发一种具有高选择性和灵敏性的分析方法来检测Cys是有很大研究价值与意义的。荧光生物成像技术支撑的可视化研究，在生命分域中扮演非常重要的角色。利用荧光探针高灵敏度、可控开关操作、选择性好、响应时间短等优点，易实现实时原位检测和观察。
Yoon等公开了一种用基于加成反应的Cys荧光探针CyAC(结构见式1’，Yoon.etal.Chem.Sci.,2012,3,2760-2765)，与Cys作用之后探针的荧光强度出现并增强，从而实现检测Cys。Yang等公开了一种用于检测Cys和Hcy的荧光探针1(结构见2’，Anal.Chem.2014,86,1800-1807)，与Cys和Hcy作用后探针的荧光增强从而检测Cys和Hcy的存在。但是，上述探针的合成方法复杂，产率低，耗时耗力。而且这类荧光探针激发发射波长短，检测信号受检测背景干扰严重。不能区分Cys和Hcy等硫醇，在精确检测环境、细胞和活体内的Cys时存在很大的应用局限。因此，开发易于合成、具有良好选择性，激发发射波长处于近红外荧光区的Cys荧光探针是环境和生命体系中分析研究Cys必不可少的工具。
发明内容
本发明目的在于提供一种易于合成、具有良好选择性，激发发射波长处于近红外荧光区的检测半胱氨酸的荧光探针化合物及其应用。
为实现上述目的，本发明采用技术方案为：
一种检测半胱氨酸的荧光探针化合物，化合物为基于花菁的荧光探针化合物，结构式如式Ⅰ所示，
本发明还提供一种检测半胱氨酸的荧光探针化合物的应用，所述式Ⅰ所示化合物在检测环境、生理条件下、细胞以及活体内的Cys中的应用。
进一步的，所述式Ⅰ所示化合物作为检测Cys荧光探针在细胞中的应用。
本发明的有益效果：
本发明化合物可作为用于选择性检测模拟生理环境和细胞内Cys的荧光探针，此探针可以选择性地与Cys反应，反应后的荧光强度发生明显改变，从而实现对Cys的检测；同时，探针对GSH、Hcy、其他多种氨基酸、H2S、活性氧、离子等物质没有显著的荧光变化。
附图说明
图1为本发明实施例提供的荧光探针对Cys的选择性；
图2为本发明实施例提供的探针在加入不同浓度Cys后的紫外吸收光谱图；
图3为本发明实施例提供的荧光探针BCy-AC荧光强度随Cys浓度变化的示意图；
图4为本发明实施例提供的荧光探针BCy-AC对HT-22细胞的细胞毒性研究；
图5为本发明实施例提供的荧光探针BCy-AC用于检测HT-22细胞内内源性和外源性添加Cys的共聚焦显微镜照片。
具体实施方式
下面结合附图及实施例用于进一步说明本发明，但本发明不限于实施例。
实施例1
式Ⅰ化合物(探针BCy-AC的合成)合成路线图如下：
在10毫升乙腈中加入1,1,2-三甲基-1H-苯并[e]吲哚(105.0mg,0.5mmol)与碘乙烷(78.0mg,0.5mmol)，将混合物加热回流12小时，过滤后所得固体物质与4-羟基异苯甲醛(30.0mg,0.2mmol)在正丁醇和甲苯的混合溶剂(7:3,v/v)中加热回流3小时即可得到绿色的粗产物(式II所示化合物)。用乙酸乙酯/甲醇(3:1,v/v)作为洗脱液在硅胶层析柱(200-300目)上分离纯化所得粗产物，产率79％。
取250mL圆底烧瓶，向其中加入10mL二氯甲烷，之后将丙烯酰氯(0.16mL,2mmol)和少量N,N-二异丙基乙胺加入，用氩气保护0℃下搅拌30分钟，用分液漏斗将10mL二氯甲烷和式II(177mg，0.3mmol)逐滴加入到圆底烧瓶中，0℃搅拌1小时后常温搅拌过夜。二氯甲烷萃取后有机相旋干，硅胶柱纯化得到式Ⅰ所示的化合物BCy-AC，产率64.1％。
1H NMR(500MHz,CDCl3-d1)δ(ppm):8.10-8.09(m,1H),7.94-7.93(m,2H),7.87-7.83(m,4H),7.72-7.71(m,3H),7.54-7.52(m,6H),7.37-7.35(m,2H),7.23-7.22(s,2H),5.36-5.34(t,2H),3.90-3.86(q,4H),1.33-1.25(m,18H).13C NMR(125MHz,CDCl3-d1)δ(ppm):182.20,167.77,166.12,139.06,132.35,131.17,130.95,130.47,129.95,129.68,129.49,128.95,128.87,128.18,127.44,126.94,123.42,122.13,109.84,65.61,45.81,23.99,13.76.LC-MS(ESI+):m/z C45H44N2O2 2+calcd.322.1696,found[M2+]322.5638。
BCy-AC对半胱氨酸的选择性：
于10ml比色管中加入BCy-AC，再加入10mM HEPES pH 7.4到5ml，摇匀，然后加入各种待测物，最后用pH 7.4的HEPES定容到10mL，探针终浓度为10.0μM。BCy-AC对Cys的选择性如图1所示,待测物工作液依次为2倍量的：1,Cys；2,Hcy；3,GSH(1mM)；4,H2S；5,Gly；6,Ser；7,Glu；8,Lys；9,Ala；10,H2O2；11,ClO-；12,Ca2+；13,Fe2+；14,Cu2+；15,Zn2+。摇匀溶液，平衡10min，将上述溶液倒进荧光皿测定荧光光谱(参见图1)。由图1的实验结果可以得出，式Ⅰ化合物BCy-AC作为荧光探针对Cys具有理想的选择性响应，能不受其他生物硫醇、活性生物分子和离子等干扰。因此，此化合物式Ⅰ在生理pH条件下能对半胱氨酸有很好的选择性。
BCy-AC对半胱氨酸的光谱响应：
测定荧光光谱的条件为：
加入式Ⅰ化合物BCy-AC分别加入到10ml比色管中，用10mM HEPES(pH7.4)稀释溶液(终浓度为10.0μM)，将不同浓度的Cys(终浓度为0-20μM)分别加入到各个比色管中。摇匀溶液平衡10分钟后取1mL于紫外吸收皿中测定各溶液400-800nm波长处的紫外吸收光谱，如图2所示。上述式Ⅰ化合物对不同浓度的Cys有不同程度的紫外吸收改变，随着Cys浓度的增加探针的紫外吸收有明显增强。由图2可见BCy-AC对半胱氨酸具有很好的紫外吸收选择性和规律性。在上述相同的测试条件下，每组样品取1mL于荧光皿测定荧光光谱(参见图3)，与Cys作用后，BCy-AC对应的726nm荧光显著改变。由图3可见Cys的加入引起体系式I化合物和式II化合物比例的改变，Cys与化合物BCy-AC相互反应后在726nm处的荧光光谱逐渐增强，表明随着Cys浓度的增加，探针的荧光发射有明显变化。由图2和图3的实验结果表明，BCy-AC对Cys具有很好的选择性，适合用于细胞以及生物体的研究。
BCy-AC对细胞生物兼容性的实验验证：
采用3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐(MTT)法检测探针的生物安全性。在96孔板上培养HT-22细胞(106细胞/毫升)。然后将不同浓度的探针BCy-AC溶液(0-80μM)分别添加到多组细胞中，对照组的细胞仅加入溶剂，培养24小时。PBS清洗后，加入MTT溶液(5.0mg/mL,20μL，PBS)。孵育4小时后，去除剩余的MTT，加入DMSO溶解甲臜晶体。用酶标仪在490nm波长处测定其光吸收值
应用例1BCy-AC用于细胞外源性半胱氨酸的检测：
用10μM BCy-AC孵育正常的HT-22细胞15min作为对照组，在荧光726nm处的成像结果，如图5a所示；
加入300μM的Cys，以提供外源性半胱氨酸。在培养箱中孵育30分钟，用DMEM清洗3遍，随后加入10μM BCy-AC，再在培养箱中孵育10min，再用DMEM清洗后进行共聚焦成像，如图5b所示。
如图5a所示，细胞几乎没有荧光；图5b所示，细胞内荧光显著增强。可见BCy-AC能够用于检测细胞中外源性的Cys。
所述荧光探针定性、定量检测Cys具体为：
将式Ⅰ应用于检测Cys时，其是与被检测物Cys作用后，生成具有式II结构的化合物，从而导致荧光强度的改变；
用HEPES缓冲溶液配置Cys浓度呈梯度变化的水溶液，分别向浓度梯度变化的Cys溶液中加入式Ⅰ的HEPES缓冲溶液。而后分别测定体系中加入Cys前后的荧光强度，然后以Cys溶液的浓度为横坐标、最大发射波长处的荧光强度值为纵坐标作图，根据荧光强度值，即可从图中读出溶液中Cys的含量。
本发明化合物采用花菁染料式II作为荧光母体，该荧光团具有高量子产率、近红外发射波长，可以最大限度地提高组织穿透力，同时使血红蛋白和肌红蛋白、水和脂质中血红素的吸收最小化。近红外荧光探针由其较低的能量可以减弱对生物体的光损伤，提高生物兼容性，更适合于实时成像Cys在细胞和体内的分布。考虑到吸电子基团能猝灭荧光团的荧光，选择含有双亲电位点的丙烯酸酯作为光诱导电子转移过程的调制基团，这能够高选择性和高灵敏性的响应Cys。通过引入酯键将荧光团与识别配体连接。通过光诱导电子转移过程来操纵荧光发射以实现选择性地检测Cys。首先Cys上的巯基与探针分子上的丙烯酸酯结构发生亲核加成反应后产物是式II的烯醇式结构，随后这个产物经过分子内烯醇式-酮式互变异构转化成具有显著荧光的式II结构，从而实现荧光开启式的检测Cys。利用检测到Cys后释放荧光团前后导致化合物荧光性质的改变，对整个化合物荧光强度的影响作为识别的检测信号，并将化合物用于检测细胞内Cys的荧光成像。
本发明化合物如结构式Ⅰ所示，以所述化合物作为Cys选择性响应的荧光探针。本发明提供了一种可用于选择性检测溶液、细胞内Cys的荧光探针,在Cys存在下，探针BCy-AC的荧光强度在726纳米处发生明显从而增强实现Cys的检测，并可大大降低外部检测条件的干扰，提高检测精度。
本发明化合物用作为Cys荧光探针，在Cys存在下荧光强度发生变化，可用于Cys的定性和定量检测。尤其是，这种化合物用作荧光探针，可用于细胞、组织以及活体中Cys的检测，细胞毒性实验(如图4所示)也证实了不同浓度探针BCy-AC孵育HT-22细胞后都没有显著的细胞毒性。这类化合物作为荧光探针可用于细胞内外Cys水平的检测，这对深入研究Cys在生物体内的产生、转运及累积等过程的动力学机理，对进一步了解Cys的生理和病理作用具有重要意义。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。作为荧光染料是本发明新化合物的一种用途，不能认定本发明的化合物仅用于荧光染料，对于本发明所属技术领域的普通技术人员来说，在基于本发明化合物用作荧光染料的相同作用机理的考虑下，还可以做出若干简单推理，得出本发明的化合物的其他应用用途，都应当视为属于本发明的保护范围。
Claims (2)
1.一种检测半胱氨酸的荧光探针化合物，其特征在于：所述化合物为基于花菁的荧光探针化合物，结构如式Ⅰ所示，
2.一种如权利要求1所述的化合物在制备检测半胱氨酸的荧光探针中的应用。
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