CN108358906A - 一种特异性区分不同硫醇的荧光探针 - Google Patents
一种特异性区分不同硫醇的荧光探针 Download PDFInfo
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Abstract
本发明涉及一种特异性区分不同硫醇的荧光探针，属于荧光探针领域。其分子结构如下：该探针分子没有荧光，与苯硫酚响应后溶液发蓝光、红光，与GSH响应后溶液发蓝光，与Hcy/Cys响应后溶液发蓝光、绿光。本发明所述的探针分子不仅可以区分不同种类的硫醇，而且可以实现快速、定量检测不同种类的硫醇，在生物化学、环境监测等领域具有重要的应用价值。
Description
技术领域
本发明涉及一种特异性区分不同硫醇的荧光探针的制备及其在细胞内检测生物硫醇与苯硫酚的应用，属于荧光探针领域。
背景技术
硫醇是生物硫醇和苯硫酚的总称，在生物体内、化学化工行业是一类重要的化合物。生物硫醇包括半胱氨酸(Cys)、同型半胱氨酸(Hcy)和谷胱甘肽(GSH)，在生物体内中发挥着重要作用。半胱氨酸缺乏会导致肝损伤，生长迟缓，皮肤病变，脱发和昏睡；血液中高浓度的同型半胱氨酸会引起心血管疾病、精神疾病和阿尔茨海默病；谷胱甘肽在生物体内含量是最丰富的，维持细胞功能、保持细胞内稳态、细胞内信号转导、异物代谢和基因调控。苯硫酚是有机合成的关键反应中间体，可广泛应用于药物和农药的生产。但苯硫酚毒性更大，鱼的半数致死剂量(LC50)在0.01mM到0.4mM之间。研究表明，长时间暴露于苯硫酚可能会导致咳嗽，恶心，呕吐，头痛，烧灼感甚至死亡。科学家们一致致力于研发一种可以在生物体内实现选择性区分Cys/Hcy、GSH和苯硫酚的简单高效的方法。
荧光探针具有选择性高、灵敏度高、实时性强、操作简单等优点，在生物检测方面具有很大的优势。近些年来基于各种硫醇巯基的亲核性强弱不同原理设计了一系列检测生物硫醇、苯硫酚的荧光探针。但由于生物硫醇和苯硫酚之间的化学性质相似，亲核性也相似，导致这些探针中的大多数选择性不好。研究表明在生理条件下，苯硫酚比生物硫醇具有更强的亲核性，不能选择性区分苯硫酚和生物硫醇。但是关于同时区分和检测半胱氨酸/同型半胱氨酸、谷胱甘肽和苯硫酚的荧光探针的报道资料不多。
发明内容
本发明目的之一是提供区分硫醇的荧光探针；目的之二是提供一种灵敏度高、选择性好、抗干扰能力强能够在细胞内区分不同硫醇的荧光探针的应用。
本发明解决问题采取的技术方案为，一种特异性区分不同硫醇的荧光探针，其分子结构式如下：具体合成路线如下：
具体成方法如下:(1)将化合物1(500mg，2.6mmol)溶于15mL甲苯中，再加入氰基乙酸(440mg，5.2mmol)，回流反应3h，停止反应。反应液冷却到室温，抽滤，滤饼用二氯甲烷洗涤，得到490mg米黄色粉末为化合物2，产率73.1％。(2)将化合物3(102mg，0.25mmol)、哌啶(20μL)溶于7.5mL二氯甲烷，将化合物2(97mg，0.37mmol)溶于7.5mL乙醇，混合两种溶液。氩气保护45℃反应10h。反应结束后，旋干溶剂，余物柱层析分离提纯，先用二氯甲烷、乙酸乙酯作为洗脱剂(V:V＝15:1)，再用二氯甲烷、甲醇作为洗脱剂(V:V＝15:1)进行柱层析，得到50mg红色粉末为化合物CQ，产率30.1％。(3)将化合物CQ(64mg，0.1mmol)溶于10mL丙酮，再加入NBD-Cl(40mg，0.2mmol)、碳酸钾(60mg，0.4mmol)，室温搅拌3h。停止反应，旋干溶剂，余物用柱层析分离纯化，用二氯甲烷、乙酸乙酯(V:V＝20:1)作为洗脱剂，得到21mg紫红色粉末为探针NCQ，产率26.4％。
本发明的荧光探针测试方法如下，将探针分子溶解在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中，室温下进行测试。可以不同种类硫醇进行定量检测，具体实施方法在实施实例中详细介绍。
探针分子的响应过程如：由于2,4-二硝基的PET(光诱导电子转移)效应，探针NCQ不发光，苯硫酚与探针NCQ作用后释放出发红光的染料QC、发蓝光的化合物1；GSH与探针NCQ作用后产生发蓝光的CQ；Cys与探针NCQ作用后产生发蓝光的CQ、发绿光的NBD-N-Cys；Hcy与探针NCQ作用后产生发蓝光的CQ、发绿光的NBD-N-Hcy，具体响应情况如下：
本发明的荧光探针在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中不发光，与苯硫酚响应后溶液的发射峰在490nm、624nm，与GSH响应后溶液的发射峰在490nm，与Hcy/Cys响应后溶液的发射峰在490nm、544nm。
本发明的荧光探针在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中不发光，与苯硫酚响应后溶液发蓝光、红光，与GSH响应后溶液发蓝光，与Hcy/Cys响应后溶液发蓝光、绿光。
本发明的荧光探与苯硫酚、GSH、Cys/Hcy作用，响应速度很快，在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中探针与苯硫酚、GSH、Cys、Hcy分别在15min、5min、6min、10min完全响应。
本发明的荧光探具有很好的灵敏度，探针(10μM)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中与苯硫酚、Cys、Hcy、GSH完全响应的浓度分别为70μM、15μM、30μM、10μM。
本发明的荧光探针与苯硫酚、GSH、Cys/Hcy作用，具有很多好的选择性、抗干扰性。探针(10μM)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中加入1mM的Ala、Ph、Met、Gly、Glu、Arg、Lys、Tyr、Leu、Pro,、Trp、Ser、PO4 3-、S2O3 2-、SCN-、SO4 2-、Cl-、CO3 2-、NO2 -、SO3 2-、NO3 -、AcO-、N3 -、I-、Na+、K+、Ca2+、Mg2+、H2O2、ClO-等，溶液的荧光光谱几乎与纯探针溶液的荧光光谱一致。
本发明的荧光探针在生理pH环境中可以实现苯硫酚、GSH、Cys/Hcy定性、定量检测。
本发明所述的探针分子合成路线简单，操作简单，成本较低，可以实现快速区分检测各种硫醇。
附图说明
图1为本发明荧光探针(10μM)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中与70μM苯硫酚、15μM Cys、30μM Hcy、10μM GSH作用后溶液的荧光光谱。横坐标为波长，纵坐标为荧光强度。
图2为本发明荧光探针(10μM)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中不同浓度Cys作用后溶液的荧光光谱，激发波长：423nm。横坐标为波长，纵坐标为荧光强度。
图3为本发明荧光探针(10μM)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中不同浓度Cys作用后溶液在490nm、544nm处的荧光强度。横坐标为Cys浓度，纵坐标为荧光强度。
图4为本发明荧光探针(10μM)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中不同浓度Cys作用后溶液在490nm、544nm处的荧光强度与Cys浓度之间的线性拟合关系。横坐标为Cys浓度，纵坐标为荧光强度。
图5为本发明荧光探针(10μM)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中不同浓度Hcy作用后溶液的荧光光谱，激发波长：423nm。横坐标为波长，纵坐标为荧光强度。
图6为本发明荧光探针(10μM)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中不同浓度Hcy作用后溶液在490nm、544nm处的荧光强度。横坐标为Hcy浓度，纵坐标为荧光强度。
图7为本发明荧光探针(10μM)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中不同浓度Hcy作用后溶液在490nm、544nm处的荧光强度与Hcy浓度之间的线性拟合关系。横坐标为Hcy浓度，纵坐标为荧光强度。
图8为本发明荧光探针(10μM)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中不同浓度GSH作用后溶液的荧光光谱，激发波长：423nm。横坐标为波长，纵坐标为荧光强度。
图9为本发明荧光探针(10μM)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中不同浓度GSH作用后溶液在490nm处的荧光强度。横坐标为GSH浓度，纵坐标为荧光强度。
图10为本发明荧光探针(10μM)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中不同浓度GSH作用后溶液在490nm处的荧光强度与GSH浓度之间的线性拟合关系。横坐标为GSH浓度，纵坐标为荧光强度。
图11为本发明荧光探针(10μM)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中不同浓度苯硫酚作用后溶液的荧光光谱，激发波长：432nm。横坐标为波长，纵坐标为荧光强度。
图12为本发明荧光探针(10μM)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中不同浓度苯硫酚作用后溶液在490nm、624nm处的荧光强度。横坐标为苯硫酚浓度，纵坐标为荧光强度。
图13为本发明荧光探针(10μM)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中不同浓度苯硫酚作用后溶液在490nm、624nm处的荧光强度与苯硫酚浓度之间的线性拟合关系。横坐标为苯硫酚浓度，纵坐标为荧光强度。
图14为本发明荧光探针(10μM)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中与15μM的Cys作用后溶液在490nm、544nm处的荧光强度与时间的关系。横坐标为时间，纵坐标为荧光强度。
图15为本发明荧光探针(10μM)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中与30μM的Hcy作用后溶液在490nm、544nm处的荧光强度与时间的关系。横坐标为时间，纵坐标为荧光强度。
图16为本发明荧光探针(10μM)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中与10μM的GSH作用后溶液在490nm处的荧光强度与时间的关系。横坐标为时间，纵坐标为荧光强度。
图17为本发明荧光探针(10μM)在pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7:3)中与70μM的苯硫酚作用后溶液在624nm、490nm处的荧光强度与时间的关系。横坐标为时间，纵坐标为荧光强度。
图18为本发明荧光探针(10μM)在活细胞(HeLa)中与各种硫醇作用后细胞成像图。
具体实施实例
实施例1：中间产物2的合成
将化合物1(500mg，2.6mmol)溶于15mL甲苯中，再加入氰基乙酸(440mg，5.2mmol)，回流反应3h，停止反应。反应液冷却到室温，抽滤，滤饼用二氯甲烷洗涤，得到490mg米黄色粉末为化合物2，产率73.1％。HRMS(ESI)m/z:理论值C13H8NO5[M-H]+,258.0408；实验值,258.0420。1H NMR(400MHz,DMSO)δ7.59(d,J＝8.7Hz,1H),6.81(dd,J＝8.7,2.1Hz,1H),6.76(d,J＝2.1Hz,1H),6.31(s,1H),5.44(s,2H),4.23(s,2H),3.86(s,1H)。13C NMR(101MHz,DMSO)δ166.2,164.5,161.9,160.6,155.5,150.1,126.5,113.6,109.4,108.6,102.9,63.2,25.0。
实施例2：化合物CQ的合成
将化合物3(102mg，0.25mmol)、哌啶(20μL)溶于7.5mL二氯甲烷，将化合物2(97mg，0.37mmol)溶于7.5mL乙醇，混合两种溶液。氩气保护45℃反应10h。反应结束后，旋干溶剂，余物柱层析分离提纯，先用二氯甲烷、乙酸乙酯作为洗脱剂(V:V＝15:1)，再用二氯甲烷、甲醇作为洗脱剂(V:V＝15:1)进行柱层析，得到50mg红色粉末为化合物CQ，产率30.1％。HRMS(ESI)m/z:理论值C32H26N5O10[M-H]+,640.1680；实验值,640.1666。1H NMR(400MHz,DMSO)δ10.61(s,1H),8.83(d,J＝2.4Hz,1H),8.42(dd,J＝9.3,2.4Hz,1H),8.14(s,1H),7.62(s,1H),7.52(d,J＝9.3Hz,1H),7.14(d,J＝9.3Hz,1H),6.82–6.67(m,2H),6.61(s,1H),6.12(s,1H),5.42(s,2H),3.59(s,2H),3.46–3.39(m,2H),1.23(s,4H),1.18(t,J＝7.0Hz,3H),1.06(t,J＝6.9Hz,3H)。13C NMR(101MHz,DMSO)δ163.3,161.9,160.5,156.1,155.5,150.5,150.0,145.3,144.3,141.6,138.9,133.0,130.3,126.4,122.3,118.6,118.0,113.5,110.5,109.5,108.6,106.6,102.9,101.9,91.3,62.9,47.6,45.9,45.3,44.3,11.2,9.9。
实施例3：探针的合成
将化合物CQ(64mg，0.1mmol)溶于10mL丙酮，再加入NBD-Cl(40mg，0.2mmol)、碳酸钾(60mg，0.4mmol)，室温搅拌3h。停止反应，旋干溶剂，余物用柱层析分离纯化，用二氯甲烷、乙酸乙酯(V:V＝20:1)作为洗脱剂，得到21mg紫红色粉末为探针NCQ，产率26.4％。HRMS(ESI)m/z:理论值C38H27N8O13[M-H]+,803.1698；实验值,803.4059。1H NMR(400MHz,DMSO)δ8.86(d,J＝2.8Hz,1H),8.67(d,J＝8.3Hz,1H),8.43(dd,J＝9.3,2.8Hz,1H),8.17(s,1H),7.93(d,J＝8.8Hz,1H),7.65–7.57(m,2H),7.40(dd,J＝8.8,2.4Hz,1H),7.16(d,J＝9.3Hz,1H),7.03(d,J＝8.3Hz,1H),6.63(s,1H),6.42(s,1H),5.54(s,2H),3.59(d,J＝4.9Hz,2H),3.43(dd,J＝14.1,7.0Hz,2H),1.31–1.22(m,4H),1.18(t,J＝7.0Hz,3H),1.06(t,J＝7.0Hz,3H)。13C NMR(101MHz,DMSO)δ163.3,161.9,160.5,156.1,155.5,150.5,150.0,145.3,144.3,141.6,138.9,133.0,130.3,126.4,122.3,118.7,118.0,113.5,110.5,109.5,108.6,106.6,102.9,101.9,91.4,62.9,47.6,45.9,45.3,44.3,34.1,31.8,29.5,29.2,22.6,14.4,11.2,10.0。
实施例3：本发明荧光探针的应用
将本发明探针溶于pH＝7.4PBS(10.0mM)与乙腈混合溶液(V:V＝7：3)中,配制成1.0×10-5mol/L的探针溶液，加入不同的硫醇进行荧光光谱测试，加入Cys/Hcy溶液在490nm、544nm处有两个荧光发射峰，加入GSH溶液在490nm处有一个发射峰，加入苯硫酚溶液在490nm、624nm处有两个发射峰。在测试体系中探针与苯硫酚、GSH、Cys、Hcy分别在15min、5min、6min、10min完全响应。本发明的荧光可以在细胞内区分、检测不同硫醇。
Claims (3)
1.一种特异性区分不同硫醇的荧光探针，其化学结构式为：
2.根据权利要求1所述的特异性区分不同硫醇的荧光探针的合成方法，其特征在于按照以下步骤进行合成：
(a)将二羟基香豆素衍生物溶于甲苯，再加入氰基乙酸，回流反应3h，反应液冷却到室温，抽滤，滤饼用二氯甲烷洗涤，得到纯产物氰基乙酸香豆素衍生物；
(b)将氰基乙酸香豆素衍生物、哌啶溶于二氯甲烷，将化合物2溶于乙醇，混合两种溶液，氩气保护45℃反应10h，旋干溶剂，柱层析分离提纯，先用二氯甲烷、乙酸乙酯作为洗脱剂，再用二氯甲烷、甲醇作为洗脱剂进行柱层析，得到喹喔啉氰基乙酸香豆素衍生物。
3.根据权利要求1所述的特异性区分不同硫醇的荧光探针，其区分特征在于探针与苯硫酚响应后溶液发蓝光、红光，与GSH响应后溶液发蓝光，与Hcy/Cys响应后溶液发蓝光、绿光。
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