CN113735890A - 可避免细胞内GSH干扰的Cys荧光探针及制备和应用 - Google Patents
可避免细胞内GSH干扰的Cys荧光探针及制备和应用 Download PDFInfo
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Abstract
本发明涉及荧光探针领域，具体涉及一种可避免细胞内GSH干扰的Cys荧光探针及制备和应用。近年来一系列高选择性Cys荧光探针被相继开发，这些探针尽管在一定程度上排除了GSH引发的荧光信号的干扰，但GSH消耗探针所带来的敏感性降低的限制仍然没有有效地克服。为解决上述问题，本发明开发了一种不受GSH干扰的特异性Cys荧光探针及其制备和应用，该荧光探针不仅能直接与Cys反应产生大的荧光关‑开响应，而且其与GSH的反应产物能进一步被Cys取代，产生同样的荧光产物和荧光响应，因此不仅能避免细胞内高浓度GSH对荧光信号的干扰，而且能避免GSH对探针的消耗。
Description
技术领域
本发明涉及荧光探针领域，具体涉及一种可避免细胞内GSH干扰的Cys荧光探针及制备和应用。
背景技术
线粒体呼吸作用除了为细胞的生存与发展提供能量之外，也会因为呼吸链的电子泄露诱发大量的活性氧(ROS)，主要包括超氧自由基(O2 ·-)、过氧化氢(H2O2)、次氯酸(HClO)、次溴酸(HBrO)、羟基自由基(HO·)。在细胞氧化还原平衡失衡的条件下，过度产生的ROS能够氧化细胞内各种生物大分子，造成细胞正常功能的损伤，最终引发各种各样的疾病。这种倾向于氧化一方的氧化与抗氧化失衡被称为氧化应激(Oxidative Stress)。实际上，为了维持氧化还原平衡，细胞天然表达了各种各样的抗氧系统，其中小分子生物硫醇构成了细胞内重要的抗氧系统之一，在维持细胞内氧化还原平衡方面扮演了关键的角色。其中，半胱氨酸(Cys)是一个重要的生物硫醇。在生物体内，Cys与同型半胱氨酸(Hcy)、谷胱甘肽(GSH)共同维持着生物体内氧化-还原平衡；其次，由于Cys结构中巯基的强亲核能力和配位能力，Cys能与有毒的芳香族化合物缩合成硫醚氨酸，还能与铜、汞等重金属络合，从而起到解毒作用。另外，Cys还参与多种蛋白质的构成，同时还与生物催化、蛋白质的翻译后修饰等生物反应有关。由于这些重要的生理功能，Cys在体内含量的异常将导致严重的疾病发生。
鉴于此，开发一种生物兼容的、选择性高且敏感的Cys检测方法，不仅对于Cys的各种已知的和未知的生理病理功能的研究，而且对于相关治疗药物的开发均意义重大。
在各种检测方法中，荧光探针技术由于分析时间短、操作简单、灵敏度高、可视化以及样品的无损性等特点，已是现代生物医学研究不可缺少的工具，在揭示生物活性分子、离子的定位及功能方面发挥了巨大的作用。鉴于此，近十几年来，利用生物硫醇发生的系列特异性化学反应，大量的生物硫醇荧光探针被相继报道。然而，由于Cys与GSH有相似的结构和反应性且细胞内Cys浓度(200-300μM)远低于GSH的浓度(1-10mM)，开发能避免细胞内GSH干扰的Cys荧光探针具有相当的挑战。需要指出的是，尽管同型半胱氨酸(Hcy)与Cys的结构和反应性及其相似(仅差一个-CH2-基团)，由于其极低的细胞内浓度(～10μM)，其所造成的干扰通常可以忽略不计。迄今报道的能选择性传感细胞内Cys的荧光探针主要分为如下三类:醛基成环型、加成-环化型和取代-重排型。尽管基于上述三个策略的荧光探针能高选择性荧光传感Cys，但在实际使用时仍存在一些缺点。例如，大部分“醛基成环型”Cys荧光探针的反应性弱、水溶性低，导致了敏感性差、荧光响应时间长、生物兼容性低，而且该类探针的荧光调控机理不明确，设计时难以把握荧光响应的方式；“加成-环化型”和“取代-重排型”Cys荧光探针尽管能避免GSH引发的荧光信号的干扰，但其能被细胞内高浓度的GSH所消耗，导致细胞内探针浓度的降低，因此影响了细胞内Cys的检测敏感性。因此，如何避免这些缺点，尤其是GSH消耗探针的问题，是当前设计Cys荧光探针所面临的重要挑战。硅吡啰红是一类具有高的摩尔消光系数和荧光量子产率、强的pH耐受性、良好的水溶性和光稳定性的近红外荧光染料。重要的是，硅吡啰红及其衍生物的9号碳原子具有强的电正性，能与亲核试剂发生加成或芳香亲核取代反应。利用该性质，针对上述挑战，本发明利用取代反应的可逆性和重排反应的不可逆性，合成了一种可避免GSH干扰的Cys荧光探针SiPyCl。
发明内容
针对上述问题，本发明提供了一种可避免GSH干扰的Cys荧光探针及其制备、应用。该探针可与Cys发生“取代-重排”反应，生成一个红荧光的“胺基-硅吡啰红”染料，而与GSH仅发生取代反应，生成一个非荧光的“硫代硅吡啰红”染料。重要的是，SiPyCl与GSH反应后生成的“硫代硅吡啰红”染料能进一步与Cys发生“取代-重排”反应，生成红荧光的“胺基-硅吡啰红”；而且，该反应的效率很高，甚至能在毫摩尔水平GSH的存在下几分钟内完成。因此，无论GSH是否存在，探针SiPyCl均能与Cys反应生成红荧光的“胺基-硅吡啰红”染料。换句话说，SiPyCl是一个可避免GSH干扰的Cys荧光探针。
为了达到上述目的，本发明采用了下列技术方案：
一种可避免细胞内GSH干扰的Cys荧光探针，所述探针为SiPyCl，其结构式为：
一种可避免细胞内GSH干扰的Cys荧光探针的制备方法，包括以下步骤：
(1)在氮气保护下，将4，4’-亚甲基双(N，N-二甲基苯胺)(化合物1)溶于超干四氢呋喃中，控温-78℃条件下，向上述反应液中逐滴滴入正丁基锂，继续控温反应2小时；在该温度下，向上述反应液中逐渐滴入二氯二甲基硅烷，反应液升至室温并搅拌反应2小时，反应结束后，加入盐酸水溶液以中和反应液，蒸干四氢呋喃，残余液体用乙醚萃取，合并的有机相分别用饱和NaHCO3溶液、水、饱和氯化钠水溶液洗涤，干燥旋干后得二氢硅吡啰红(化合物2)，化合物2无需进行提纯直接进行下一步反应；
(2)将二氢硅吡啰红(化合物2)溶于丙酮中，在冰盐浴下条件控温-15℃，向上述溶液中加入高锰酸钾粉末，反应液恢复到室温继续反应2小时，反应液经过滤、干燥和柱色谱分离后得到硅吡啰红酮(化合物3)为黄色固体；
(3)将硅吡啰红酮(化合物3)溶解在干燥的二氯甲烷中，向上述溶液中逐滴滴加草酰氯，反应液室温搅拌反应10分钟，反应结束后，将溶剂旋干，粗产品经柱色谱分离得所述探针SiPyCl。
进一步，所述步骤(1)中4，4’-亚甲基双(N，N-二甲基苯胺)、正丁基锂与二氯二甲基硅烷的摩尔比为1：4：1.8。
进一步，所述步骤(1)中盐酸水溶液的浓度为1mol/L。
进一步，所述步骤(3)中硅吡啰红酮与草酰氯的摩尔比为1:1.2。
进一步，所述步骤(3)中柱色谱分离展开剂CH2Cl2：CH3CN的体积比为5:2。
一种可避免细胞内GSH干扰的Cys荧光探针的应用，在制备检测细胞内Cys试剂中的应用。
与现有技术相比本发明具有以下优点：
GSH在细胞中的浓度为毫摩尔(1-10mM)水平，目前报道的大部分Cys荧光探针存在易被细胞内GSH消耗的限制。本发明提供的探针能直接与Cys发生“取代-重排”反应生成高荧光的“胺基-吡啰红”染料，而且其与GSH反应生成的非荧光的“硫代吡啰红”染料能进一步与Cys发生“取代-重排”反应生成高荧光的“胺基-吡啰红”染料。因此本发明提供的探针是一个能避免细胞内高浓度GSH消耗探针的高选择性Cys荧光探针。
附图说明
图1为化合物3的1H NMR图(CDCl3,600MHz)；
图2为化合物3的13C NMR图(CDCl3,150MHz)；
图3为化合物3的HRMS图；
图4为化合物SiPyCl的1H NMR图(CDCl3,600MHz)；
图5为化合物SiPyCl的13C NMR图(CDCl3,150MHz)；
图6为化合物SiPyCl的HRMS图；
图7为探针在PBS中荧光光谱随时间(0-30min)的变化图。
图8为探针SiPyCl与Cys/GSH反应所引起的吸收和荧光光谱变化，(图8A)Cys/GSH引发的SiPyCl的吸收光谱变化；(图8B)Cys/GSH引发的SiPyCl的荧光光谱变化；(图8C)在GSH缺乏下，SiPyCl对Cys的荧光滴定光谱；(图8D)在GSH(1mM)存在下SiPyCl对Cys的荧光滴定光谱；(图8E)探针SiPyCl对Cys的选择性结果；
图9为本发明探针SiPyC1对Cys的传感机理示意图。
具体实施方式
实施例1
一种可避免细胞内GSH干扰的Cys荧光探针，其结构式为：
一种所述可避免细胞内GSH干扰的Cys荧光探针的制备方法，包括以下步骤：
(1)氮气保护下，将化合物1(6.00g，14.6mmol)溶于超干四氢呋喃(200mL)中，控温-78℃条件下，向上述反应液中缓慢滴入正丁基锂(浓度为2.4M的正己烷溶液，24.3mL，58.4mmol，4当量)，继续控温反应2小时；在该温度下，向上述反应液中缓慢滴入二氯二甲基硅烷(3.2mL，26.28mmol，1.8当量)，反应液缓慢升至室温并搅拌反应2小时；反应结束后，小心加入盐酸水溶液(1M，50mL)以中和反应液；蒸干四氢呋喃，残余液体用乙醚萃取，合并的有机相分别用饱和NaHCO3溶液、水、饱和氯化钠水溶液洗涤，干燥旋干后得化合物2，化合物2无需进行提纯直接进行下一步反应。
(2)将化合物2溶于丙酮(30mL)中，在冰盐浴下控温-15℃条件下，向上述溶液中缓慢加入高锰酸钾粉末(5.75g)，反应液恢复到室温继续反应2小时。反应液经过滤、干燥和柱色谱(CH2Cl2)分离后得到化合物3为黄色固体(1.65g，产率34.7％)。
1H NMR(600Hz,CDCl3)δ8.10(d,J＝9.0Hz,2H),6.87(d,J＝9.0Hz,2H),6.83(s,2H),3.11(s,12H),0.49(s,6H).13C NMR(150MHz,CDCl3)δ185.3,151.4,140.5,131.6,129.7,114.3,113.2,40.1,0.97.ESI-MS:[M+H]+calcd for 325.1736,Found 325.1734.
(3)将化合物3(0.324g，1.0mmol)溶解在干燥的二氯甲烷(10mL)中，向上述溶液中缓慢滴加草酰氯(1.2mmol，1.2当量)，反应液室温搅拌反应10分钟。反应结束后，将溶剂旋干，粗产品经柱色谱(CH2Cl2/CH3CN＝5/2，v/v)分离得探针SiPyC1(0.313g，产率82.8％)。
1H NMR(600Hz,CDCl3)δ8.43(d,J＝9.6Hz,2H),7.14(s,2H),6.97(d,J＝9.6Hz,2H),3.48(s,12H),0.58(s,6H).13C NMR(150MHz,CDCl3)δ164.0,155.6,148.7,140.9,127.4,121.4,119.4,42.7,0.41.ESI-MS:[M]+calcd for 343.1392,Found 343.1390.
实施例2
1.测试溶液配制
将探针用乙腈配成2mM的储存液，随后用20mM的PBS(pH 7.4)稀释至测试浓度。
2.探针稳定性实验
首先在荧光光谱仪上研究探针在水溶液中的稳定性，配置探针的PBS溶液(2μM)，置于比色皿中，如图7所示，探针在PBS中的发射波长为688nm，连续扫描30分钟后，荧光光谱几乎保持不变。上述结果表明，阳离子特性赋予了探针优良的水溶性且可以在PBS中稳定存在，该特性对于生物应用至关重要。
3.探针与Cys/GSH的反应性能研究
在PBS缓冲中，SiPyCl本身在663nm处有一个主要的吸收峰；加入Cys后，一个蓝移的吸收峰出现在470nm处，而加入GSH后，一个红移的吸收峰出现在688nm处。该结果暗示了SiPyCl分别与Cys和GSH生成了“胺基-硅吡啰红”和“硫代吡啰红”染料(图8A)。在470nm的激发光激发下，SiPyCl本身几乎是非荧光的；加入Cys后，一个强的荧光峰出现在618nm处；加入GSH后，几乎没有任何荧光峰被观察(图8A)。该结果暗示了SiPyCl能避免GSH引发的干扰荧光信号。重要的是，当在SiPyCl溶液中首先加入GSH然后再加入Cys后，GSH所引起的红移的吸收峰消失，而Cys引起的蓝移的吸收峰出现，而且这个蓝移的吸收峰的吸收强度与GSH缺乏下的情况几乎一致(图8A)。不仅如此，当在SiPyCl溶液中首先加入GSH然后再加入Cys后，一个强的荧光峰也被观察，而且该峰的荧光密度与GSH缺乏下的情况也几乎一致(图8B)。因此，SiPyCl也是一个能避免GSH消耗探针的Cys荧光探针。进一步的荧光滴定化验表明，在缺乏(图8C)和存在(图8D)1mM的GSH的情况下，SiPyC1几乎表现了一致的Cys传感性能，而且后者对低浓度的Cys表现了更好的荧光响应。选择性实验表明，生物相关的阳离子、阴离子、活性氧化物、氨基酸均不会引发SiPyCl的荧光增强(图8E)，因此探针对Cys具有非常好的选择性。
4.探针对Cys的传感机理研究
在纯PBS中，SiPyCl能与Cys发生快速的“取代-重排”反应生成高荧光的胺基-硅吡啰红(λex:470nm)；然而，由于不稳定的大环过渡态，SiPyCl与GSH仅能发生“取代”反应而生成在470nm激发下非荧光的硫代硅吡啰红；重要的是，硫代硅吡啰红能进一步与Cys发生快速的“取代-重排”反应，同样生成高荧光的胺基-吡啰红；因此，SiR是一个可避免GSH干扰的(包括信号干扰和消耗探针)、高敏感的Cys荧光探针。发明人推测，Cys能取代SiR-GSH中GSH单元的推动力来自于两方面：一是Si-吡啰红9号碳原子大的电正性，二是Cys取代GSH单元后随即发生的不可逆重排，前者建立了一个Cys/GSH与探针之间的平衡反应，后者促使了总反应向右的不可逆移动。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，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 (7)
1.一种可避免细胞内GSH干扰的Cys荧光探针，其特征在于，所述探针的结构式为：
2.一种权利要求1所述可避免细胞内GSH干扰的Cys荧光探针的制备方法，其特征在于，包括以下步骤：
(1)在氮气保护下，将4，4’-亚甲基双(N，N-二甲基苯胺)溶于超干四氢呋喃中，控温-78℃条件下，向上述反应液中逐滴滴入正丁基锂，继续控温反应2小时；在该温度下，向上述反应液中逐渐滴入二氯二甲基硅烷，反应液升至室温并搅拌反应2小时，反应结束后，加入盐酸水溶液以中和反应液，蒸干四氢呋喃，残余液体用乙醚萃取，合并的有机相分别用饱和NaHCO3溶液、水、饱和氯化钠水溶液洗涤，干燥旋干后得二氢硅吡啰红；
(2)将二氢硅吡啰红溶于丙酮中，在冰盐浴下条件控温-15℃，向上述溶液中加入高锰酸钾粉末，反应液恢复到室温继续反应2小时，反应液经过滤、干燥和柱色谱分离后得到硅吡啰红酮；
(3)将硅吡啰红酮溶解在干燥的二氯甲烷中，向上述溶液中逐滴滴加草酰氯，反应液室温搅拌反应10分钟，反应结束后，将溶剂旋干，粗产品经柱色谱分离得所述探针。
3.根据权利要求2所述的一种可避免细胞内GSH干扰的Cys荧光探针的制备方法，其特征在于，所述步骤(1)中4，4’-亚甲基双(N，N-二甲基苯胺)、正丁基锂与二氯二甲基硅烷的摩尔比为1：4：1.8。
4.根据权利要求2所述的一种可避免细胞内GSH干扰的Cys荧光探针的制备方法，其特征在于，所述步骤(1)中盐酸水溶液的浓度为1mol/L。
5.根据权利要求2所述的一种可避免细胞内GSH干扰的Cys荧光探针的制备方法，其特征在于，所述步骤(3)中硅吡啰红酮与草酰氯的摩尔比为1:1.2。
6.根据权利要求2所述的一种可避免细胞内GSH干扰的Cys荧光探针的制备方法，其特征在于，所述步骤(3)中柱色谱分离展开剂CH2Cl2：CH3CN的体积比为5:2。
7.一种权利要求1所述可避免细胞内GSH干扰的Cys荧光探针的应用，其特征在于，在制备检测细胞内Cys试剂中的应用。
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