CN114436947B - 一种对粘度和硝基还原酶双响应的荧光探针及其制备方法和应用 - Google Patents
一种对粘度和硝基还原酶双响应的荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于有机小分子荧光探针技术领域，具体涉及一种对粘度和硝基还原酶双响应的荧光探针及其制备方法和应用。本发明的荧光探针通过加入无水DMF和叔丁醇钾，氮气下，回流，冷却至室温，将反应液倒入冰水中，过滤，洗涤，干燥得到化合物1；加入无水乙腈、化合物1和对硝基溴化苄，在N2保护下回流，冷却至室温，过滤，用柱层析法纯化，制得探针。所述探针对溶液中粘度变化和硝基还原酶的检测显著敏感，随着溶液粘度的增大，探针在610nm处的荧光强度显著增强；在NADPH参与下，随着硝基还原酶浓度增大，探针M440被硝基还原酶还原为化合物1，其在520nm处的荧光显著增强；经细胞实验表明所述探针能很好的定位在线粒体，检测细胞中线粒体粘度的变化以及缺氧条件下硝基还原酶的产生。
Description
技术领域
本发明属于有机小分子荧光探针技术领域，具体涉及一种对粘度和硝基还原酶双响应的荧光探针及其制备方法和应用。
背景技术
现有技术公开了细胞中线粒体粘度是胞内生物分子间物质交换,信号传递和相互作用的重要影响因素，粘度异常是判断细胞恶性肿瘤、阿尔兹海默病、糖尿病和动脉粥样硬化等疾病的参考指标。研究公开了当细胞中氧气浓度不足时会导致细胞缺氧，而缺氧是肿瘤组织的特征之一，有研究表明细胞或者组织在缺氧状态下会过表达硝基还原酶，硝基还原酶可以作为常见的肿瘤组织缺氧的标志物，因此，精确监测活细胞线粒体内粘度和细胞缺氧时硝基还原酶的变化具有重要的意义。
研究显示了小分子荧光探针由于其高灵敏度，实时分析，无创检测等优点，在细胞内微环境检测方面具有广泛的应用；但是，目前能够定位线粒体同时进行粘度和硝基还原酶检测的荧光探针几乎未见有报道，因此，开发新型的能够双响应粘度和硝基还原酶的荧光探针，具有良好的应用价值。
基于现有技术的现状。本申请的发明人拟提供一种对粘度和硝基还原酶双响应的荧光探针及其制备方法和应用。
发明内容
本发明的目的在于基于现有技术的现状，提供一种对粘度和硝基还原酶双响应的荧光探针及其制备方法和应用。
具体的，本发明提供了一种合成简便、靶向精确的对粘度和硝基还原酶双响应的荧光探针及其制备方法。
本发明提出了对粘度和硝基还原酶双响应的荧光探针，所述荧光探针的结构式如下，简称：M440；
所述的对粘度和硝基还原酶双响应的荧光探针通过下述方法制备，其包括步骤：
(1)制备化合物1：在50mL的圆底烧瓶中加入10.0mL，130.4mmol的无水DMF和2.4g，21.3mmol的叔丁醇钾，氮气保护下，60℃回流搅拌反应7小时，然后冷却至室温，将反应液倒入冰水中，有黄色固体析出，过滤，然后用甲醇/水(V：V＝1：1)洗三遍，干燥后得到黄色固体化合物1；
(2)合成制备探针M440:在50mL两口烧瓶里加入20.0mL无水乙腈、1.122g，5.0mmol的化合物1和1.082g，5.0mmol的对硝基溴化苄，再在N2保护下85℃回流搅拌12小时；然后停止反应并冷却至室温，过滤后用石油醚洗三遍得到粗产物，将粗产物使用柱层析法纯化，制得探针M440。
本发明制得的探针对溶液中粘度变化和硝基还原酶的检测非常敏感，随着溶液粘度的增大，探针在610nm处的荧光强度显著增强；而且在NADPH的参与下，随着硝基还原酶的浓度增大，探针M440会被硝基还原酶还原为化合物1，其在520nm处的荧光显著增强；经细胞实验表明，本发明的探针能很好的定位在线粒体，并且可以检测细胞中线粒体粘度的变化以及缺氧条件下硝基还原酶的产生。
进一步，本发明提供了所述的荧光探针在检测细胞线粒体粘度和硝基还原酶中的应用。
本发明所述的荧光探针可用于制备检测细胞线粒体粘度和硝基还原酶产品，优选的，所述产品为检测与细胞线粒体粘度和硝基还原酶相关疾病的试剂盒。
本发明所述的试剂盒中，检测粘度的溶液体系为甲醇-甘油体系，检测硝基还原酶的溶液体系为三羟甲基氨基甲烷盐酸盐(Tris-HCl)体系。
本发明中，所述的与细胞线粒体粘度和硝基还原酶相关疾病选自恶性肿瘤、糖尿病、动脉粥样硬化和阿尔茨海默病中的一种或多种。
本发明的有益效果如下：
本发明所述的荧光探针是一类检测细胞内线粒体粘度和硝基还原酶变化的荧光探针分子，该探针合成简单，易于应用。该探针能够实现在溶液中对粘度进行检测，还能在NADPH的存在下对硝基还原酶响应，并且实现细胞内对线粒体共定位成像，在细胞内进行粘度和硝基还原酶的双响应，该探针在荧光生物成像领域具有潜在的应用价值。
附图说明
图1为荧光探针M440的核磁共振氢谱图；
图2为荧光探针M440的核磁共振碳谱图；
图3为荧光探针M440在不同粘度体系中的荧光强度；
图4为荧光探针M440在不同浓度的硝基还原酶体系中的荧光强度；
图5为荧光探针M440与商品化染料Mito-Tracker Green的细胞共定位图像；
图6为荧光探针M440检测细胞MHCC-97H内粘度变化的图像；
图7为荧光探针M440检测细胞MHCC-97H缺氧时硝基还原酶变化的图像。
具体实施方式
下面结合实施例对本发明作进一步阐述，但这些实施例绝不是对本发明的任何限制。
实施例1：探针M440的合成制备
合成化合物1：在50mL的圆底烧瓶中加入无水DMF(10.0mL，130.4mmol)和叔丁醇钾(2.4g，21.3mmol)，氮气保护下，60℃回流搅拌反应7小时，然后冷却至室温，将反应液倒入冰水中，有黄色固体析出，过滤，然后用甲醇/水(V：V＝1：1)洗三遍，干燥后得到黄色固体化合物1。1HNMR(400MHz,DMSO-d6)δ8.50(d,J＝4.9Hz,2H),7.44(d,J＝8.7Hz,2H),7.34(d,J＝5.9Hz,2H),7.26(d,J＝16.3Hz,1H),6.80(d,J＝16.2Hz,1H),6.71(d,J＝8.8Hz,2H),3.03(s,6H).13C NMR(100MHz,DMSO-d6)δ145.51,143.77,140.95,128.66,124.69,123.08,118.70,115.26,106.79,34.91；
合成制备探针M440:在50mL两口烧瓶里加入20.0mL无水乙腈、化合物1(1.122g，5.0mmol)和对硝基溴化苄(1.082g，5.0mmol)，再在N2保护下85℃回流搅拌12小时。然后停止反应并冷却至室温，过滤之后用石油醚洗三遍得到粗产物，将粗产物使用柱层析法纯化，得探针M440，紫红色固体，产率81％。1HNMR(400MHz,DMSO-d6)δ8.86(d,J＝6.8Hz,2H),8.28(d,J＝8.8Hz,2H),8.08(d,J＝6.7Hz,2H),7.94(d,J＝16.0Hz,1H),7.71(d,J＝8.6Hz,2H),7.58(d,J＝8.6Hz,2H),7.17(d,J＝16.0Hz,1H),6.81-6.73(m,2H),5.80(s,2H),3.01(s,6H).13C NMR(100MHz,DMSO-d6)δ154.30,151.91,147.58,143.64,142.87,141.78,130.26,129.49,124.00,122.57,122.23,116.80,111.80,60.45。
实施例2：探针M440在溶液中对粘度的响应
将实施例1中的探针M440溶解于DMSO中，配制成浓度为1.0mM的母液。分别取10μL上述母液加入1mL不同体积比(甲醇:甘油＝90:10；80:20；70:30；60:40；50:50；40:60；30:70；20:80；10:90,0:100)的甲醇/甘油体系中，然后进行荧光检测，结果如图3所示，随着甘油体积比重的增加，探针的荧光强度逐渐增强，表明该探针M440可以在甘油体系中对粘度有很好的响应关系。
实施例3：探针M440在溶液中对硝基还原酶的响应
将实施例1中的探针M440配制终浓度为10μM，在500μM NADPH存在下，分别加入不同浓度(0～5.0μg/mL)的硝基还原酶在37℃充分反应60分钟后，进行荧光检测，如图4所示，可以观察到在520nm附近的发射峰荧光强度不断增强，表明该探针M440可以在溶液体系中对硝基还原酶有很好的响应关系。
实施例4：探针M440在细胞MHCC-97H中的线粒体共定位
将实施例1中的探针M440用DMSO配成1mM母液,首先弃去培养皿中的上清液，加入10μM莫能霉素对细胞预处理30分钟，然后再加入终浓度为10μM探针M440于37℃孵育30分钟，最后加入1.0μM Mito-Tracker Green，再继续孵育15分钟，接着用新鲜的PBS(10mM)洗涤细胞三次。通过共聚焦激光扫描荧光显微镜，检测探针M440是否定位于细胞线粒体。对于探针M440而言，用495nm激光作为激发光源，用黄色荧光通道(580-630nm)采集发射光谱；对于Mito-Tracker Green而言，用488nm激光作为激发光源，用绿色通道(500-550nm)采集发射光谱；共聚焦荧光显微镜图像如图5所示，显示了探针M440能很好的定位于细胞内线粒体。
实施例5：探针M440对细胞MHCC-97H内粘度变化的检测
莫能霉素可以作为离子载体诱导线粒体粘度发生改变，如图6所示，通过共聚焦激光扫描荧光显微镜，当MHCC-97H细胞中仅加入10μM探针M440孵育时，在黄色通道的荧光很弱。而先加入10μM莫能霉素对细胞预处理30分钟，再与10μM探针M440孵育30分钟，在相同的测试条件下，黄色荧光通道中细胞的荧光强度明显增强，表明该探针可以对细胞内线粒体粘度的变化进行检测。
实施例6：探针M440对细胞MHCC-97H内硝基还原酶变化的检测
缺氧条件下癌细胞中会过表达硝基还原酶。将细胞分为两组培养，一组氧气含量20％，另一组氧气含量2％，培养8小时后，分别向两组细胞中加入实施例1中的探针M440，使其终浓度为10μM，然后继续孵育30分钟，将培养皿中的培养液弃掉，用PBS洗三遍。在共聚焦激光扫描荧光显微镜成像，结果如图7所示，氧气含量为2％的实验组的细胞与探针作用后，荧光信号明显强于氧气含量为20％的实验组细胞，表明该探针能检测细胞内的硝基还原酶。
Claims (5)
1.一种对粘度和硝基还原酶双响应的荧光探针，其特征在于：所述荧光探针的结构式如下，简称：M440；
2.权利要求1的对粘度和硝基还原酶双响应的荧光探针在制备检测细胞线粒体粘度和硝基还原酶产品中的应用。
3.根据权利要求2所述的应用，其特征在于，所述产品为检测与细胞线粒体粘度和硝基还原酶相关疾病的试剂盒。
4.根据权利要求3所述的应用，其特征在于，所述试剂盒中，检测粘度的溶液体系为甲醇-甘油体系，检测硝基还原酶的溶液体系为三羟甲基氨基甲烷盐酸盐(Tris-HCl)体系。
5.根据权利要求3所述的应用，其特征在于，所述的与细胞线粒体粘度和硝基还原酶相关疾病选自恶性肿瘤、糖尿病、动脉粥样硬化和阿尔茨海默病中的一种或多种。
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