CN112939887B - 一种基于碱性染料的近红外荧光探针及其制备方法和应用 - Google Patents
一种基于碱性染料的近红外荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种基于碱性染料的近红外荧光探针及其制备方法和应用。所述探针的化合物名称为4‑((2,4‑二硝基苯)磺酰基)氧基)苄基3,7‑双(二乙氨基)‑10H‑苯恶嗪‑10‑羧酸盐，或者是4‑((2,4‑二硝基苯)磺酰基)氧基)苄基3,7‑双(二甲氨基)‑10H‑吩噻嗪‑10‑羧酸盐；探针分别命名为L1和L2。本发明还提供了基于碱性染料的近红外荧光探针L1或L2在GSH检测中的应用，即在磷酸缓冲溶液pH＝7：乙醇/9：1(v/v)体系中，通过荧光分光光度计定量检测GSH的含量，该检测方法灵敏度高，方法简便。
Description
技术领域
本发明涉及碱性染料和GSH检测，具体属于一种基于碱性染料的近红外荧光探针及其制备方法，以及该探针在检测GSH含量中的应用。
背景技术
谷胱甘肽(glutathione，GSH)在调节生命的氧化还原平衡态、维持生物的正常生命活动和疾病预防方面发挥着重要作用。但GSH浓度异常会导致癌症、阿尔茨海默氏症和心血管等疾病。因此，精准示踪这些活性分子的水平变化是深入了解细胞功能的前提，也是揭示细胞生命活动规律的本质。近红外荧光探针(650-900nm)，由于其较高的信噪比、较强的穿透深度以及较高的成像分辨率，受到了研究者的重视。其中，碱性染料近红外荧光生色团，是生物分析领域中一种常用的组织染料。其在近红外区的吸收峰强且尖锐，摩尔消光系数高。故碱性染料及其衍生物可以作为良好的构建探针的骨架。
基于此，高稳定性、高选择性、灵敏度高、具有低细胞毒性的近红外荧光探针用于检测细胞内GSH的水平变化尤为重要，将为当前生物医学发展提供一个有力的科学工具。
在本发明中，基于碱性染料荧光团合成了两个检测GSH的近红外荧光探针，探针以磺酸酯为GSH的识别基团，经过电荷转移释放出碱性染料荧光团，从而实现对GSH的检测。
发明内容
本发明的目的在于提供一种基于碱性染料的近红外荧光探针及其制备方法，以及将该探针用于GSH含量的检测，且检测过程较为简便、灵敏度较高；为深入了解生物硫醇生命体系中的具体功能提供有力工具。
本发明提供的两个基于碱性染料的近红外荧光探针，它们的化合物的名称为：
4-((2,4-二硝基苯)磺酰基)氧基)苄基3,7-双(二乙氨基)-10H-苯恶嗪-10-羧酸酯 (4-(((2,4-dinitrophenyl)sulfonyl)oxy)benzyl 3,7-bis(diethylamino)-10H-phenoxazine-10-carboxylate)；
5-4-((2,4-二硝基苯)磺酰基)氧基)苄基3,7-双(二甲氨基)-10H-吩噻嗪-10-羧酸酯 (4-(((2,4-dinitrophenyl)sulfonyl)oxy)benzyl 3,7-bis(dimethylamino)-10H-phenothiazine-10-carboxylate)；
分别命名为L1和L2，其结构式为：
本发明提供了两个基于碱性染料的近红外荧光探针的合成方法，步骤如下：
将对羟基苯甲醇溶于二氯甲烷中，于冰水浴中搅拌5min，随后加入三乙胺，搅拌30min 后滴加溶于二氯甲烷的2，4-二硝基苯磺酰氯，搅拌反应过夜，TLC检测反应进程，用50mL 饱和氯化钠溶液分别萃取三次，用50mL二氯甲烷溶液分别萃取三次，无水硫酸钠干燥有机相，用石油醚/乙酸乙酯＝3：1过柱纯化得纯产物4-(羟甲基)苯基2,4-二硝基苯磺酸酯。上述所用原料对羟基苯甲醇：三乙胺：2,4-二硝基苯磺酰氯摩尔比为1：3：1。
将4-(羟甲基)苯基2,4-二硝基苯磺酸酯 ，3,7-双(二乙氨基)-10H-吩恶嗪-10-羰基氯，碳酸钠和4-二甲氨基吡啶溶于二氯甲烷中，在氮气保护，冰浴条件下搅拌反应8h。除去溶剂并将粗产物进行硅胶柱色谱纯化，得到棕色固体4-((2,4-二硝基苯)磺酰基)氧基)苄基3,7-双(二乙氨基)-10H-苯恶嗪-10-羧酸酯 (命名为L1)。其中4-(羟甲基)苯基2,4-二硝基苯磺酸酯 ：3,7-双(二乙氨基)-10H-吩恶嗪-10-羰基氯：碳酸钠：4-二甲氨基吡啶的投料摩尔比为1.2：1：3：1。
将4-(羟甲基)苯基2,4-二硝基苯磺酸酯 ，3,7-双(二甲氨基)-10H-吩噻嗪-10-羰基氯，碳酸钠和4-二甲氨基吡啶溶于二氯甲烷中，在氮气保护，冰浴条件下搅拌反应8h。除去溶剂并将粗产物进行硅胶柱色谱纯化，得到棕色固体4-((2,4-二硝基苯)磺酰基)氧基)苄基3,7-双(二甲氨基)-10H-吩噻嗪-10-羧酸酯 。(命名为L2)。其中4-(羟甲基)苯基2,4-二硝基苯磺酸酯 ：3,7- 双(二甲氨基)-10H-吩噻嗪-10-羰基氯：碳酸钠：4-二甲氨基吡啶的投料摩尔比为1.2：1：3：1。
所述基于碱性染料的近红外荧光探针L1、L2可用于对GSH的检测。
本发明提供的一种检测GSH的方法，步骤为：
(1)、配制pH＝7磷酸缓冲溶液(10mM，10％无水乙醇)为反应体系，将L1，L2溶于DMSO配制成2mM的制备液，配置20mM的谷胱甘肽(GSH)溶液。
(2)、取1900μL的反应体系溶液、2μL L1(L2)的DMSO溶液、100μL的GSH溶液，加到一个荧光比色皿中，37℃水浴下进行荧光扫描，随着时间的增加，676nm(L1)686nm (L2)处荧光强度增强。
与现有技术相比，本发明具有如下优点和效果：
1、本发碱性染料近红外荧光衍生物合成简单，成本较低；
2、本发明检测手段简单，只需紫外分光光度计和荧光检测器即可；
3、本发明近红外荧光探针L1、L2能实现对GSH的动态检测，灵敏度较高。
4、本发明检测信号明显，为近红外荧光信号，反应溶液颜色变化肉眼可见，从无色变成淡蓝色。
附图说明：
图1实施例1制备荧光探针L1的氢谱图
图2实施例1制备荧光探针L1的碳谱图
图3实施例1制备荧光探针L1的质谱图
图4实施例1制备荧光探针L2的氢谱图
图5实施例1制备荧光探针L2的碳谱图
图6实施例1制备荧光探针L2的质谱图
图7实施例2L1与GSH作用的荧光光谱图
图8实施例2L2与GSH作用的荧光光谱图
图9实施例3L1、L2在HepG-2中的荧光成像图
图10实施例3L1+GSHee在HepG-2中的荧光成像图
具体实施方式
下面结合实施例和附图对本发明做进一步说明，但本发明不受下述实施例的限制。
实施例1L1、L2的合成和表征
将对羟基苯甲醇(5mmol，620.6mg)溶于15mL二氯甲烷中，于冰水浴中搅拌5min,随后加入三乙胺(15mmol，2mL)，搅拌30min后滴加溶于10mL二氯甲烷的2，4-二硝基苯磺酰氯(5mmol，1333mg)，搅拌反应过夜，TLC检测反应进程，用50mL饱和氯化钠溶液分别萃取三次，用50mL二氯甲烷溶液分别萃取三次，无水硫酸钠干燥有机相，用石油醚/乙酸乙酯＝3：1过柱纯化得纯产物4-(羟甲基)苯基2,4-二硝基苯磺酸酯 (3.86mmol， 1367.5mg)，产率77.2％。
将4-(羟甲基)苯基2,4-二硝基苯磺酸酯 (0.6mmol，212.4mg)，3,7-双(二乙氨基)-10H- 吩恶嗪-10-羰基氯(0.5mmol，194.1mg)，碳酸钠(Na2CO3，1.5mmol，160mg)和4-二甲氨基吡啶(DMAP，0.5mmol，61.1mg)溶于20mL二氯甲烷中，在氮气保护，冰浴条件下搅拌反应8h。除去溶剂并将粗产物进行硅胶柱色谱纯化，得到棕色固体4-((2,4-二硝基苯) 磺酰基)氧基)苄基3,7-双(二乙氨基)-10H-苯恶嗪-10-羧酸酯 (0.104mmol，73.4mg)，产率20.8％。1H NMR(600MHz,DMSO)δ9.11(s,1H),8.58(d,J＝8.7Hz,1H),8.25(d,J＝8.6Hz,1H),7.45(d,J＝6.9Hz,2H),7.27(d,J＝8.9Hz,2H),7.20(d,J＝7.1Hz,2H),6.38(d,J＝9.0Hz, 2H),6.32(s,2H),5.21(s,2H),3.30(d,J＝6.6Hz,8H),1.06(t,J＝6.2Hz,12H).13CNMR(151 MHz,DMSO)δ152.93,151.57,150.80,148.15,148.08,146.12,136.67,133.60,130.81,130.24, 129.73,127.53,125.19,122.07,121.19,116.31,106.26,98.69,66.30,43.85,12.37.
将4-(羟甲基)苯基2,4-二硝基苯磺酸酯 (0.6mmol，212.4mg)，3,7-双(二甲氨基)-10H- 吩噻嗪-10-羰基氯(174.5mg，0.5mmol)，碳酸钠(Na2CO3，1.5mmol,160mg)和4-二甲氨基吡啶(DMAP，0.5mmol，61.1mg)溶于20mL二氯甲烷中，在氮气保护，冰浴条件下搅拌反应8h。除去溶剂并将粗产物进行硅胶柱色谱纯化，得到棕色固体4-((2,4-二硝基苯)磺酰基)氧基)苄基3,7-双(二甲氨基)-10H-吩噻嗪-10-羧酸酯 (0.05mmol，33.3mg)，产率10％。1H NMR(600MHz,DMSO)δ9.12(s,1H),8.58(d,J＝8.6Hz,1H),8.25(d,J＝8.6Hz,1H),7.39 (d,J＝7.9Hz,2H),7.32(d,J＝8.8Hz,2H),7.20(d,J＝8.0Hz,2H),6.68(s,2H),6.65(d,J＝8.8 Hz,2H),5.17(s,2H),2.89(s,12H).13C NMR(151MHz,DMSO)δ153.44,151.53,148.66,148.12, 147.97,136.79,133.57,132.00,130.80,129.36,127.48,127.40,126.99,121.99,121.16,110.87, 109.73,66.05,40.19.
实施例2
配制pH＝7的磷酸缓冲溶液(10mM，含无水乙醇10％)体系溶液，将L1，L2溶于DMSO配制成2mM的制备液，配置20mM的谷胱甘肽(GSH)溶液；取1900μL的反应体系溶液、 2μL L1(L2)的DMSO溶液、100μL的GSH溶液加到一个荧光比色皿中，37℃水浴下进行荧光扫描，随着时间的增加，676nm(L1)686nm(L2)处荧光强度增强。荧光发射图见图7、图8。
实施例3
配制pH＝7.4、浓度为10mM的PBS缓冲溶液，配制2μM L1、L2的DMSO溶液；10μL L1、L2的DMSO溶液加入到2mL的PBS溶液中，使得其浓度为10μM；将此溶液加入HepG-2 细胞中，在37℃下，孵育30min后，于共聚焦成像，荧光成像仪下显示较强的红色荧光；见图9。
用2mL的50μM的NEM(GSH抑制剂)溶液孵育HepG-2细胞30min，2mL PBS 洗涤三次，再加2mL10μM的探针L1(L2)孵育30min，2mL PBS洗涤三次，加入2mL PBS 溶液，进行共聚焦成像。红色荧光强度减弱；见图9。
用2mL的50μM的NEM(GSH抑制剂)溶液孵育HepG-2细胞30min，2mL PBS 洗涤三次，再分别用2mL 200μM、1mM的GSHee孵育30min，2mL PBS洗涤三次，L1 孵育30min，2mL PBS洗涤三次，加入2mL PBS溶液，进行共聚焦成像，红色荧光强度减弱；见图10。
Claims (5)
1.一种基于碱性染料的近红外荧光探针，其特征在于，结构式为L1或L2所示：
2.如权利要求1所述的基于碱性染料的近红外荧光探针的合成方法，其特征在于，步骤如下：
将对羟基苯甲醇溶于二氯甲烷中，于冰水浴中搅拌5min，随后加入三乙胺，搅拌30min后滴加溶于二氯甲烷的2,4-二硝基苯磺酰氯，搅拌反应过夜，TLC检测反应进程，用饱和氯化钠和二氯甲萃取洗涤，无水硫酸钠干燥有机相，用石油醚/乙酸乙酯＝3：1过柱纯化得纯产物4-(羟甲基)苯基2,4-二硝基苯磺酸酯 ；上述所用原料对羟基苯甲醇：三乙胺：2,4-二硝基苯磺酰氯摩尔比为1：3：1；
将4-(羟甲基)苯基2,4-二硝基苯磺酸酯 ，3,7-双(二乙氨基)-10H-吩恶嗪-10-羰基氯，碳酸钠和4-二甲氨基吡啶溶于二氯甲烷中，在氮气保护，冰浴条件下搅拌反应8h；除去溶剂并将粗产物进行硅胶柱色谱纯化，得到棕色固体4-((2,4-二硝基苯)磺酰基)氧基)苄基3,7-双(二乙氨基)-10H-苯恶嗪-10-羧酸酯 ，命名为L1；其中4-(羟甲基)苯基2,4-二硝基苯磺酸酯 ：3,7-双(二乙氨基)-10H-吩恶嗪-10-羰基氯：碳酸钠：4-二甲氨基吡啶的投料摩尔比为1.2：1：3：1；
或者，
将4-(羟甲基)苯基2,4-二硝基苯磺酸酯 ，3,7-双(二甲氨基)-10H-吩噻嗪-10-羰基氯，碳酸钠和4-二甲氨基吡啶溶于二氯甲烷中，在氮气保护，冰浴条件下搅拌反应8h；除去溶剂并将粗产物进行硅胶柱色谱纯化，得到棕色固体4-((2,4-二硝基苯)磺酰基)氧基)苄基3,7-双(二甲氨基)-10H-吩噻嗪-10-羧酸酯 ，命名为L2；其中4-(羟甲基)苯基2,4-二硝基苯磺酸酯 ：3,7-双(二甲氨基)-10H-吩噻嗪-10-羰基氯：碳酸钠：4-二甲氨基吡啶的投料摩尔比为1.2：1：3：1。
3.如权利要求1所述的一种基于碱性染料的近红外荧光探针L1或L2在GSH检测中的应用。
4.一种检测GSH的方法，包括如下步骤：
(1)、配制pH＝7、10mM的、含10％无水乙醇的磷酸缓冲溶液为反应体系，将L1或L2溶于DMSO配制成2mM的制备液，配制20mM的谷胱甘肽(GSH)溶液；
(2)、取1900μL的反应体系溶液、2μL L1或L2的DMSO溶液、100μL的GSH溶液，加到一个荧光比色皿中，37℃水浴下进行荧光扫描，随着时间的增加，676nm(L1)或686nm(L2)处荧光强度增强。
5.如权利要求1所述的探针L1或L2在制备细胞成像试剂中的应用。
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