CN108641710B - 一种检测蛋白质硫巯基化的荧光探针及其制备方法和应用 - Google Patents
一种检测蛋白质硫巯基化的荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种检测蛋白质硫巯基化的荧光探针及其制备方法和应用；该探针以七甲川花菁类荧光染料为母体，其化学结构通式如式(I)所示。首次合成的针对线粒体中的蛋白质硫巯基化进行检测的荧光探针，本发明的荧光探针稳定性好，合成简单，使用方便；只与硫巯基化的蛋白质反应，不受其他含巯基/硫的化合物或生物大分子的干扰；探针具有亚细胞靶向性，可特异性的检测线粒体中的硫巯基化蛋白质。该探针在生命科学和医学研究中具有广阔的应用前景；
Description
技术领域
本发明属于生物检测领域，涉及一种检测蛋白质硫巯基化的荧光探针及其制备方法与应用。
背景技术
蛋白质硫巯基化修饰(S-sulfhydration)是一中依赖于硫化氢(H2S)和过硫化合物(H2Sn)的可逆性蛋白翻译后修饰。H2S和H2Sn可对蛋白质中特定的半胱氨酸巯基进行修饰，发生硫巯基化，改变蛋白质构相，调节蛋白质功能，是与磷酸化和乙酰化相似的新的信号转导调控方式。蛋白质硫巯基化修饰在众多生理病理过程中发挥了关键的作用，包括自噬、氧化应激、神经传导、凋亡、炎症等。此外，已有研究表明蛋白质硫巯基化修饰的过度激活或抑制与心血管疾病、呼吸系统疾病以及神经退行性疾病密切相关。蛋白质硫巯基化修饰作为一个研究热点，越来越受到研究人员的关注，发展灵敏度高、选择性好，能“实时”动态跟踪蛋白质硫巯基化修饰的分析与检测技术是最具挑战性的前沿课题之一，也是阐明蛋白质硫巯基化修饰如何发挥生理病理作用的前提。
目前检测蛋白硫巯基化的方法主要有生物素开关法(biotin switch assay)、半胱氨酸标记法(cysteinyl labelling assay)马来酰亚胺法(maleimide assay)。这些方法均存在一定不足，包括假阳性率高、操作步骤繁琐、灵敏度有限、无法实现在体实时监测等缺点。通过大量筛选，小分子荧光探针技术能够实现与硫巯基化蛋白发生特异性的反应，灵敏度较高，有较好的生物相容性及稳定性。近年来，小分子荧光探针技术逐渐成为医学和生命科学领域不可或缺的研究手段之一。
发明内容
本发明的目的在于针对上述现有技术存在的不足提供一种检测蛋白质硫巯基化的荧光探针及其制备方法与应用；本发明提供的荧光探针本身只有微弱的荧光，但可与硫巯基化蛋白发生迅速的反应，生成强荧光产物，实现大幅的荧光增强，从而实现测定表现蛋白质硫巯基化修饰的水平和定量分析。
本发明的目的是通过以下技术方案来实现的：
第一方面，本发明提供一种荧光探针，具有式I所示结构式：
其中，R1基团为氟、氯、溴、碘、C1-C4烷基或三氟甲基，R2基团为氧或硫。
第二方面，本发明提供式I所示荧光探针的制备方法，所述方法包括如下步骤：
以二氯甲烷为溶剂，在-10～25℃条件下混合IR780衍生物和取代的肉桂酰氯升温至25～28℃进行反应，即得所述荧光探针。
具体而言：将IR780衍生物溶于5mL二氯甲烷中，于0℃下加入R1、R2取代的肉桂酰氯溶于10ml二氯甲烷，室温反应两个小时。反应液旋干，用中性三氧化二铝过柱，得到式I化合物，反应式如下：
优选的，所述IR780衍生物和取代的肉桂酰氯的摩尔比为1:0.6～1:1.8。
优选的，所述IR780衍生物是通过如下步骤制备而得：
以N,N-二甲基甲酰胺为溶剂，IR780与乙酸钠在35～90℃反应3～10小时，即得所述IR780衍生物。
优选的，所述IR780与乙酸钠的摩尔比为1:0.1～1:2。
第三方面，本发明提供式I所示的荧光探针的用途，所述荧光探针用于检测细胞水平或组织水平上的蛋白质硫巯基化修饰。
本发明以活细胞中的应用为例子，可通过以下步骤实现：将式I所示探针溶液加入到细胞培养液中孵育，孵育6分钟后，然后用磷酸盐缓冲液(pH＝7.4)洗涤去除多余的探针，观察记录细胞荧光强度。
本发明利用蛋白上的硫巯基与3-(三氟甲基)肉桂酯发生特异性反应，从而将硫巯基化蛋白质与其他未被硫巯基化修饰的蛋白质进行区分。该类探针本身没有荧光，与硫巯基化的蛋白质翻译后可生成具有强荧光的产物，因而可用于蛋白质硫巯基化的修饰程度的灵敏检测。荧光探针法不仅可用于检测细胞样本、血浆、组织匀浆中的硫巯基化修饰程度，还可用于活细胞和动物组织中硫巯基化修饰的动态检测。
与现有技术相比，本发明具有如下有益效果：
(1)反应速度快，6分钟内可完成检测；
(2)稳定性好，能够长期保存使用；
(3)灵敏度高，不与其他含巯基/硫的化合物或生物大分子反应；
(4)具有亚细胞靶向性，能够检测线粒体中的蛋白硫巯基化修饰水平；
(5)可实现活细胞和组织切片水平的蛋白硫巯基化修饰水平检测。
(6)本发明荧光探针I可检测蛋白硫巯基化的性质是其他多数类似化合物所不具备的性质。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：
图1是荧光探针I-1与不同浓度的硫巯基化的木瓜蛋白酶的反应前后的荧光变化；
图2是荧光探针I-1对硫巯基化蛋白质的选择性；
图3是荧光探针I-1检测活细胞中硫巯基化蛋白；
图4是荧光探针I-1在活细胞水平与线粒体染料共定位的结果；
图5是荧光探针I-1检测组织中硫巯基化蛋白以及与线粒体染料共定位的结果；
图6是荧光探针I-1的核磁共振氢谱；
图7是荧光探针I-1的核磁共振碳谱；
图8是荧光探针I-1的高分辨质谱。
具体实施方式
下面结合附图和实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干调整和改进。这些都属于本发明的保护范围。
实施例1
化合物I-2的合成
将IR780(1g,1.5mmol)与2g乙酸钠溶于20ml DMF，80℃反应6小时，反应液用乙酸乙酯萃取并水洗三次，有机相旋干用中性三氧化二铝过柱，二氯甲烷/甲醇＝20:1作为淋洗剂过柱得到600mg红色带有金属光泽的固体I-2，产率60％。1H NMR(400MHz,CDCl3)δ8.17(d,J＝13.2Hz,2H),7.17(dd,J＝7.0,5.6Hz,4H),6.90(t,J＝7.4Hz,2H),6.67(d,J＝8.0Hz,2H),5.46(d,J＝13.2Hz,2H),3.64(t,J＝7.4Hz,4H),2.61(t,J＝5.7Hz,4H),1.88(dd,J＝11.9,6.1Hz,2H),1.76(dd,J＝14.7,7.4Hz,4H),1.67(s,12H),1.00(t,J＝7.4Hz,6H)；13CNMR(100MHz,CDCl3)δ186.4,162.4,144.4,139.7,132.9,127.6,126.5,121.7,120.4,106.7,92.5,77.4,77.0,76.7,46.5,44.1,28.8,25.9,22.6,19.8,11.7；ESI-HRMS(m/z):[M+H]+ calculated for HQO(C36H45N2O,M+):521.3532,found 521.3388.
荧光探针I-1的制备
将IR780衍生物(100mg,0.01mmol)溶于5mL二氯甲烷中，加入3-(三氟甲基)肉桂酰氯(216mg,0.01mmol)溶于10ml二氯甲烷，0℃下加入化合物2,，室温反应2小时。反应液旋干用中性三氧化二铝过柱，二氯甲烷/甲醇＝20:1作为淋洗剂过柱得到35mg绿色带有金属光泽的固体。结构验证结果如图6,7,8所示，其中图6为本发明荧光探针I-1的核磁共振氢谱。图7为本发明荧光探针I-1的核磁共振碳谱。图8为本发明荧光探针I-1的高分辨质谱结果。具体为1H NMR(400MHz,MeOD)δ8.16(d,J＝16.1Hz,1H),8.10–7.98(m,2H),7.77(d,J＝13.8Hz,3H),7.65(t,J＝7.5Hz,1H),7.29(t,J＝8.8Hz,4H),7.23–7.07(m,5H),6.14(d,J＝14.1Hz,2H),4.02(t,J＝6.9Hz,4H),2.63(s,4H),1.96–1.88(m,2H),1.77(dd,J＝14.2,7.1Hz,4H),1.49(s,12H),0.94(t,J＝7.2Hz,6H).13C NMR(100MHz,MeOD)δ172.4,164.2,159.9,147.2,142.3,141.0,140.5,134.9,131.8,130.0,128.4,125.0,122.1,121.6,117.7,110.8,100.2,49.1,45.2,28.7,27.0,23.9,20.78,20.4,10.3.ESI-HRMS(m/z):[M]+calculated for HQO-SSH(C46H50F3N2O2 +,M+):719.3824,found 719.3660.
实施例2、荧光1探针I-1与不同浓度的硫巯基化的木瓜蛋白酶的反应前后的荧光
变化
将荧光探针少量溶解于DMSO，分别加入不同浓度的硫巯基化修饰的木瓜蛋白酶，探针的终浓度为20μM，反应10分钟后，使用520nm激发，记录溶液在最大发射波长(640nm)下的荧光强度，记录其荧光光谱，如图1所示；由图1可知，荧光探针I-1可与硫巯基化蛋白质反应，并发出荧光。
实施例3、荧光探针I-1对硫巯基化蛋白质的选择性
将DMSO溶解后的荧光探针，将探针分别加入含有或不含有硫巯基化修饰的木瓜蛋白酶的不同溶液中，从1到21分别为仅空白组、仅硫巯基化修饰的木瓜蛋白酶、SCN-、SO3 2-、S2O4 2-、S2O5 2-、S2O3 2-、S-nitroso glutathione、O2 -、H2O2、OCl-、tert-BuOOH、Ala、Arg、His、Me、Ser、Thr、Trp、Tyr、Val。分别记录其溶液的荧光强度，如图2所示；由图2可知，荧光探针I-1对硫巯基化蛋白质具有选择性。
实施例4、荧光探针I-1检测活细胞中硫巯基化蛋白
在37℃、5％CO2的条件下，将A549和BEAS-2B细胞以50,000个/mL的密度接种于细胞培养液(DMEM含10％小牛血清、青霉素/链霉素(100μg/mL))中培养。细胞生长密度为80％，胰酶消化，接种于共聚焦拍照用的腔室盖玻片上。分别对细胞进行不同的方法进行处理，改变其蛋白质硫巯基化修饰的水平，包括加入PPG(2mM)、Na2S(100μM)、Na2S(100μM)+DTT(1mM)。处理后将探针I-1溶液加入到细胞培养液中孵育，然后用磷酸盐缓冲液(pH＝7.4)洗涤去除多余的探针。用激光共聚焦显微镜进行观察、拍照，其激发波长为561nm，荧光成像范围为600-650nm，表明该探针可检测细胞水平上的蛋白质硫巯基化修饰的水平，结果如图3所示。
实施例5、荧光探针I-1在活细胞水平与线粒体染料共定位的结果
将线粒体靶向染料MitoTracker与荧光探针I-1共孵育，分别用激发波长为561nm，405nm，荧光成像范围分别为600-650nm，510-590nm，观察探针I-1与MitoTracker的荧光成像结果，明确探针I-1是否与MitoTracker的荧光位置重叠，结果表明I-1与MitoTracker的荧光重叠，I-1是线粒体靶向的荧光探针，结果如图4所示。
实施例6、荧光探针I-1检测组织中硫巯基化蛋白以及与线粒体染料共定位的结果
将取正常昆明小鼠肺组织组织器官置于冰冻切片机上，进行恒冷箱连续切片，片厚10μm，对组织进行不同的处理，HQO-SSH组：肺切片不进行后续处理；HQO-SSH+Na2S(50μM)组：肺切片与50μM Na2S孵育半小时后，烘干，封片；HQO-SSH+Na2S(100μM)组：肺切片与100μMNa2S孵育半小时后，烘干，封片；HQO-SSH+Na2S+DTT组：肺切片与100μM Na2S孵育半小时后，加入DTT(1mM)，烘干，封片。此外，另设一组，将MitoTracker与荧光探针I-1共孵育，孵育后观察I-1与MitoTracker的荧光成像是否重叠。激光共聚焦显微镜观察组织的荧光，结果表明该探针可检测组织水平上的蛋白质硫巯基化修饰的水平且具有线粒体靶向。结果如图5所示。
以上对本发明的具体实施例进行了描述。需要理解的是，本发明并不局限于上述特定实施方式，本领域技术人员可以在权利要求的范围内做出各种变形或修改，这并不影响本发明的实质内容。
Claims (6)
1.一种荧光探针，其特征在于，所述荧光探针的化学结构通式如式I所示：
其中，R1基团为氟、氯、溴、碘、C1-C4烷基或三氟甲基，R2基团为氧或硫。
2.一种根据权利要求1所述的荧光探针的制备方法，其特征在于，所述方法包括如下步骤：
以二氯甲烷为溶剂，在-10～25℃条件下混合IR780衍生物和取代的肉桂酰氯升温至25～28℃进行反应，即得所述荧光探针。
3.根据权利要求2所述的荧光探针的制备方法，其特征在于，所述IR780衍生物和取代的肉桂酰氯的摩尔比为1:0.6～1:1.8。
4.根据权利要求2所述的荧光探针的制备方法，其特征在于，所述IR780衍生物是通过如下步骤制备而得：
以N,N-二甲基甲酰胺为溶剂，IR780与乙酸钠在35～90℃反应3～10小时，即得所述IR780衍生物。
5.根据权利要求4所述的荧光探针的制备方法，其特征在于，所述IR780与乙酸钠的摩尔比为1:0.1～1:2。
6.一种根据权利要求1所述的荧光探针的应用，其特征在于，所述荧光探针用于检测细胞水平或组织水平上的蛋白质硫巯基化修饰，所述应用是非疾病诊断且非疾病治疗的。
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