CN107602502B - 一种用于生物硫醇检测的esipt型荧光探针及应用 - Google Patents
一种用于生物硫醇检测的esipt型荧光探针及应用 Download PDFInfo
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Abstract
本发明公布了一种用于生物硫醇检测的ESIPT型荧光探针,为2‑(4‑(2,4‑二硝基苯基磺酰氧基)‑3‑甲酰基苯基)‑4‑甲基噻唑‑5‑羧酸乙酯;其中2,4‑二硝基苯磺酰基作为识别基团,2‑(3‑甲酰基‑4‑羟基苯基)‑4‑甲基噻唑‑2‑甲酸乙酯为信息报告基团。ESIPT型荧光探针制备方法简单,可简单迅速进入细胞中并与细胞内生物硫醇特异性结合,使其有明显的荧光增强效应,可肉眼分辨,其对常见生物分子的抗干扰能力很强,具有十分高效的选择性,可通过紫外吸收和荧光分光光度法进行分析。ESIPT荧光探针稳定性好,能够长期保存使用,适用于各种活细胞的生长环境,可实现对细胞内微量生物硫醇高灵敏度的检测,并且能够应用于细胞和活体成像,具有非常重要的应用价值。
Description
技术领域
本发明属于有机小分子荧光探针领域,涉及一种以苯基噻唑为荧光母体的荧光探针,尤其涉及以2-(3-甲酰基-4-羟基苯基)-4-甲基噻唑-2-甲酸乙酯为荧光母体的生物巯基的荧光探针及其应用。
背景技术
半胱氨酸(Cys),同型半胱氨酸(Hcy)和谷胱甘肽(GSH)是细胞中非常重要的三种巯基氨基酸,也称生物硫醇。半胱氨酸(Cys)在细胞内的含量为30-200 μM,同型半胱氨酸(Hcy)为5.0-13.9 μM和谷胱甘肽(GSH)为1-10 mM。他们在生物系统的分子和生理过程中起关键作用。例如保持氨基硫醇的稳态,生物催化,金属结合,翻译后修饰和异种生物的解毒。最近这些生物素受到很多研究的关注。氨基硫醇水平的变化与各种疾病有关。Cys与神经毒性,儿童生长缓慢,肝损伤,脂肪减少,皮肤损伤和肌肉无力密切相关。Hcy缺乏导致炎症性肠病,阿尔茨海默病和骨质疏松症的风险。GSH也与癌症,帕金森病和阿尔茨海默病等疾病直接相关。由于其重要的生物学作用,发展一种快速、灵敏、简便的检测生物硫醇的方法对于学术研究和潜在的疾病诊断非常重要。
检测生物硫醇的传统方法有高效液相色谱法、质谱法、气相色谱法及电化学方法等。而这些方法普遍存在操作复杂、仪器昂贵、检测时间长等缺点。但荧光探针由于其具有高灵敏度、操作简单、检测时间短、低成本,以及能够实现对细胞和活体的实时可视化示踪的优点,被广泛应用于生物硫醇的检测。近年来,一些反应型硫醇探针相继被报道,但荧光母体大都采用香豆素类、BODIPY、丹磺酰氮丙啶类等探针,这些探针由于本身具有较强的荧光,用于活细胞成像时会造成较低的信噪比,很少能够用于在斑马鱼体内进行荧光成像。因此,开发出能够用于检测活细胞和活体中生物硫醇的荧光探针成为亟待解决的问题。
激发式分子内质子转移(ESIPT)的荧光染料由于其高选择性,高灵敏度,低检测限,易于使用以及用荧光探针进行生物成像的巨大潜力,因此荧光检测更为理想。在不同类型的荧光探针的开发中,比率荧光探针由于来自两个不同波长的发射强度的比率而引起越来越多的关注,这可以提供环境效应的内在校正并增加荧光测量的动态范围。并且由于超快反应速率和极大荧光斯托克斯位移等显着特性的优点,ESIPT化合物在光学领域的潜在应用引起了人们的关注。但利用其检测生物硫醇报道较少。
鉴于此,ESIPT化合物作为检测生物硫醇的荧光母体结构,同时利用2,4-二硝基苯磺酰基修饰ESIPT化合物,降低自身的荧光,借此提高信噪比,有望开发出能够检测活性生物体中的生物硫醇并可以应用于活体荧光成像的新型荧光探针。
发明内容
为了提高活细胞和活体成像的信噪比,进一步开发ESIPT染料类探针,实现在各种干扰下对溶液中、活细胞和斑马鱼体内的生物巯基的检测,本发明提供一种用于生物硫醇检测的ESIPT型荧光探针。
一种用于生物硫醇检测的ESIPT型荧光探针为2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯(NL-S);
其中2,4-二硝基苯磺酰基作为识别基团,2-(3-甲酰基-4-羟基苯基)-4-甲基噻唑-2-甲酸乙酯为信息报告基团;
式(І)为所述ESIPT型荧光探针的化学结构式。
本发明ESIPT型荧光探针(NL-S)可以与生物硫醇发生亲核反应,释放出具有荧光的2-(3-甲酰基-4-羟基苯基)-4-甲基噻唑-2-甲酸乙酯,然后2-(3-甲酰基-4-羟基苯基)-4-甲基噻唑-2-甲酸乙酯再继续与Cys、Hcy反应形成席夫碱,分别得到荧光更强的2-(5-(5-(乙氧基羰基)噻唑-2-基)-2-羟基苯基)-1,3-噻二烷-4-羧酸以及2-(5-(5-(乙氧基羰基)噻唑-2-基)-2-羟基苯基)噻唑烷-4-羧酸,从而实现对生物硫醇的检测,见图1。
进一步限定的技术方案如下:
制备本发明ESIPT型荧光探针的方法如下:
将含有2-(3-甲酰基-4-羟基苯基)-4-甲基噻唑-2-甲酸乙酯(NL,1.03mmol,300mg)和三乙胺(0.5mL)的20mL无水二氯甲烷加入到三口烧瓶中,在冰浴条件下,0.5小时内向混合物中逐渐滴加2,4-二硝基苯磺酰氯(1.24mmol,330mg)的二氯甲烷(10mL)溶液,混合物在0℃下搅拌1小时。然后将混合物放在室温下搅拌反应2小时,用薄层层板监测反应结束。有机相用水(3×20 mL)洗涤,然后用无水硫酸镁干燥。减压浓缩溶剂。粗产品通过硅胶柱色谱纯化,得到荧光探针,为黄色固体。
用于4-羟乙基哌嗪乙磺酸(HEPES)/二甲基亚砜(DMSO)缓冲液中生物硫醇检测时,用所述缓冲液和ESIPT型荧光探针配制成浓度10 μM的荧光探针工作液。
用于人体肿瘤细胞HeLa细胞中生物硫醇检测时,用4-羟乙基哌嗪乙磺酸(HEPES)/二甲基亚砜(DMSO)缓冲液和ESIPT型荧光探针配制成浓度20 μM的荧光探针工作液。
用于斑马鱼中生物硫醇检测时,用4-羟乙基哌嗪乙磺酸(HEPES)/二甲基亚砜(DMSO)缓冲液和ESIPT型荧光探针配制成浓度20 μM的荧光探针工作液。
用于滤纸上生物硫醇检测时,用二氯甲烷将ESIPT型荧光探针配制成浓度10 μM的荧光探针工作液。
本发明ESIPT型荧光探针可用于化学体系中生物硫醇的检测,生物活细胞和斑马鱼体内的生物硫醇的分析检测和荧光成像检测。
配置本发明ESIPT型荧光探针的HEPES/DMSO缓冲液(pH=7.4,v/v=2:8)的溶液,分别定量加入微量的半胱氨酸(Cys),高半胱氨酸(Hcy),谷胱甘肽(GSH),脯氨酸(proline),天冬氨酸(aspartic acid),色氨酸(tryptophan),精氨酸(arginine),酪氨酸(tyrosine),组氨酸(histidine), 谷氨酸(glutamic acid),赖氨酸(lysine),苏氨酸(threonine),甘氨酸(glycine),硝酸钾(KNO3),硝酸钙(Ca(NO3)2),硝酸钠(NaNO3),硝酸镁(Mg(NO3)2),硝酸铜(Cu(NO3)2),硝酸锌(Zn(NO3)2),硝酸铁(Fe(NO3)3),双氧水(H2O2)和葡萄糖的水溶液。通过荧光分光光度法测试对不同氨基酸、人体常见金属离子、双氧水和葡萄糖的选择性和响应能力,结果见图1-4。
在不加入NEM和加入NEM的活HeLa细胞中加入终浓度为20 μM 本发明ESIPT型荧光探针的缓冲溶液,对照细胞染色后荧光显微成像变化,结果见图5。
将本发明的ESIPT型荧光探针配制成20 μM HEPES/DMSO缓冲溶液,与活体斑马鱼进行孵育,对照斑马鱼染色后共聚焦成像变化,结果见图6-7。
配制半胱氨酸(Cys),高半胱氨酸(Hcy),谷胱甘肽(GSH),脯氨酸(proline),天冬氨酸(aspartic acid),色氨酸(tryptophan),精氨酸(arginine),酪氨酸(tyrosine),组氨酸(histidine), 谷氨酸(glutamic acid),赖氨酸(lysine),苏氨酸(threonine),甘氨酸(glycine),硝酸钾(KNO3),硝酸钙(Ca(NO3)2),硝酸钠(NaNO3),硝酸镁(Mg(NO3)2),硝酸铜(Cu(NO3)2),硝酸锌(Zn(NO3)2),硝酸铁(Fe(NO3)3),双氧水(H2O2)和葡萄糖的水溶液,分别将滤纸浸入到每种溶液中。晾干之后,在滤纸上滴加10 μM本发明ESIPT型荧光探针的缓冲溶液,在365nm UV灯下,观察其颜色变化,结果见图8。
本发明的有益技术效果体现在以下方面:
1.本发明合成步骤简单,易于操作。
2.稳定性好,易于长期保存。
3.其对常见生物分子的抗干扰能力很强,适用于各种活细胞的生长环境。
4.能够在溶液中特异性检测生物硫醇,荧光强度增强27~44倍。
5.能够在溶液中高灵敏检测生物硫醇,检测限分别为Cys:0.107 μM,Hcy:0.154 μM GSH:0.206 μM。
6.可实现对细胞内微量生物硫醇高灵敏度的检测。
7.可简单进入活细胞或者活组织中,能够对生物体内的生物硫醇进行特异性识别,可适用于活细胞和活体的荧光成像。
附图说明
图1为2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯的HEPES/DMSO缓冲液(pH=7.4, v/v=2:8)中加入不同的氨基酸、人体常见金属离子、双氧水和葡萄糖对巯基类化合物的荧光发射图谱。
图2为2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯与半胱氨酸反应环化产物的高效液相质谱图。
图3为2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯与高半胱氨酸反应环化产物的高效液相质谱图。
图4为2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯与谷胱甘肽反应产物的质谱图。
图5为2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯在HeLa细胞中的荧光显微成像图。
其中:A组是HeLa细胞;B组是在37℃下,HeLa细胞用20μM探针2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯处理0.5小时;C组是HeLa细胞与1mMN-乙基马来酰亚胺(NEM)在细胞培养液中孵育2小时,PBS缓冲液洗涤三次,然后用20μM的2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯孵育0.5小时;D组是HeLa细胞与1mM NEM在细胞培养液中孵育2小时,PBS缓冲液洗涤三次,然后用20μM的2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯孵育0.5小时,再用200μM Cys孵育0.5小时。
图6为3龄斑马鱼(A组)的荧光共焦图。
图7为加入2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯,与3龄斑马鱼进行孵育(B组)的荧光共焦图。
图8为2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯与不同的氨基酸、人体常见金属离子、双氧水和葡萄糖在滤纸上进行反应的UV图。
具体实施方式
为了使本领域的技术人员更好的理解本申请的技术方案,下面将结合本申请实施例中的附图,对本申请实施例中的技术方案进行清楚、完整的描述。
下面结合实施例,对本发明作进一步的描述。
实施例1
制备用于生物硫醇检测的ESIPT型荧光探针
ESIPT型荧光探针为2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯(NL-S)。由2-(3-甲酰基-4-羟基苯基)-4-甲基噻唑-2-甲酸乙酯与2,4-二硝基苯磺酰氯反应得到2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯,生成的2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯(NL)可以与生物硫醇发生反应脱去2,4-二硝基苯基磺酰氧基释放出具有荧光的2-(3-甲酰基-4-羟基苯基)-4-甲基噻唑-2-甲酸乙酯,然后2-(3-甲酰基-4-羟基苯基)-4-甲基噻唑-2-甲酸乙酯再继续与半胱氨酸(Cys)、高半胱氨酸(Hcy)反应形成席夫碱,分别得到荧光更强的2-(5-(5-(乙氧基羰基)噻唑-2-基)-2-羟基苯基)-1,3-噻二烷-4-羧酸以及2-(5-(5-(乙氧基羰基)噻唑-2-基)-2-羟基苯基)噻唑烷-4-羧酸,从而达到检测的效果。如图1-4所示。
上述的反应过程反应式如下:
上述反应过程的具体步骤如下:
将含有2-(3-甲酰基-4-羟基苯基)-4-甲基噻唑-2-甲酸乙酯(NL,1.03mmol,300mg)和三乙胺(0.5mL)的20mL无水二氯甲烷加入到三口烧瓶中,在冰浴条件下, 0.5小时内向混合物中逐渐滴加2,4-二硝基苯磺酰氯(1.24mmol,330mg)的二氯甲烷(10mL)溶液,混合物在0℃下搅拌1小时。然后将混合物放在室温下搅拌反应2小时,用薄层层板监测反应结束。有机相用水(3×20 mL)洗涤,然后用无水硫酸镁干燥。减压浓缩溶剂。粗产品通过硅胶柱色谱纯化,得到黄色固体,熔点:185.4-186.0℃,产率36.9%。
核磁共振氢谱测定:1H NMR (600 MHz, DMSO-d 6 ): δ 10.24 (s, 1H), 8.94 (s,1H), 8.54 (s, 1H), 8.49 (m, 1H), 8.35 (m, 1H), 7.45 (m, 2H), 4.31 (m, 2H),2.71 (m, 3H), 1.30 (m, 3H)。
核磁共振碳谱测定:13C NMR (150 MHz, DMSO-d 6 ):δ189.20,167.04,161.68,160.86, 157.09,154.08,143.00,140.36,134.52,130.41,130.26,129.24,128.03,122.66,122.51, 122.31,121.47,61.83,17.65,14.57。
高分辨质谱:m/z [M+Na]+ calcd for [C20H15N3O10S2 + Na]+: 544.0091, found:544.0090。
实施例2
用于溶液体系中生物硫醇的检测
用于4-羟乙基哌嗪乙磺酸(HEPES)/二甲基亚砜(DMSO)缓冲液中生物硫醇检测时,用4-羟乙基哌嗪乙磺酸(HEPES)/二甲基亚砜(DMSO)缓冲液和ESIPT型荧光探针配制成浓度10 μM的荧光探针工作液;具体操作过程如下:
用移液枪向3mL配好的2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯的HEPES/DMSO缓冲液(pH=7.4,v/v=2:8,10μM)的溶液中,分别定量加入200μM的半胱氨酸(Cys),高半胱氨酸(Hcy),谷胱甘肽(GSH),脯氨酸(proline),天冬氨酸(aspartic acid),色氨酸(tryptophan),精氨酸(arginine),酪氨酸(tyrosine),组氨酸(histidine), 谷氨酸(glutamic acid),赖氨酸(lysine),苏氨酸(threonine),甘氨酸(glycine),硝酸钾(KNO3),硝酸钙(Ca(NO3)2),硝酸钠(NaNO3),硝酸镁(Mg(NO3)2),硝酸铜(Cu(NO3)2),硝酸锌(Zn(NO3)2),硝酸铁(Fe(NO3)3),双氧水(H2O2)和葡萄糖的水溶液,使其作用30min后进行荧光分光光度法测试,显示探针2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯对含生物硫醇具有较好的选择性,加入生物硫醇前后对照显示具有明显的荧光增强效应。见图1所示。
实施例3
细胞内荧光成像检测
用于人体肿瘤细胞HeLa细胞中生物硫醇检测时,用4-羟乙基哌嗪乙磺酸(HEPES)/二甲基亚砜(DMSO)缓冲液和ESIPT型荧光探针配制成浓度20 μM的荧光探针工作液;具体操作过程如下:
取A,B,C,D四组,A组:HeLa细胞;B组:在37℃下,HeLa细胞用20μM探针2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯处理0.5小时;C组:HeLa细胞与1mM N-乙基马来酰亚胺(NEM)在细胞培养液中孵育2小时,PBS缓冲液洗涤三次,然后用20 μM的2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯溶液孵育0.5小时;D组是HeLa细胞与1mM NEM在细胞培养液中孵育2小时,PBS缓冲液洗涤三次,然后用20μM的2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯孵育0.5小时,再用200μM Cys孵育0.5小时。荧光成像显示2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯能够渗透进细胞内。A组HeLa细胞自身无荧光。B组细胞内显较强荧光。C组不显荧光。D组显示荧光恢复。见图5所示。
实施例4
斑马鱼的荧光共聚焦成像
用于斑马鱼中生物硫醇检测时,用4-羟乙基哌嗪乙磺酸(HEPES)/二甲基亚砜(DMSO)缓冲液和ESIPT型荧光探针配制成浓度20 μM的荧光探针工作液;具体操作过程如下:
取A,B两组对照,A组:3龄斑马鱼;B组:在28℃下,用20μM探针2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯处理3龄斑马鱼0.5小时。斑马鱼的活体成像显示在28℃下用2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯(20μM)孵育0.5小时的3日龄斑马鱼在斑马鱼中显示出强烈的蓝色荧光。相反,当斑马鱼不孵育时,不能观察到荧光,结果见图6-7。荧光共聚焦成像显示2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯能够进入斑马鱼体内。A组斑马鱼自身无荧光。B组斑马鱼体内显示较强荧光。见图6和7所示。
实施例5
滤纸显色实验
用于滤纸上生物硫醇检测时,用二氯甲烷和ESIPT型荧光探针配制成浓度10 μM的荧光探针工作液;具体操作过程如下:
配制半胱氨酸(Cys),高半胱氨酸(Hcy),谷胱甘肽(GSH),脯氨酸(proline),天冬氨酸(aspartic acid),色氨酸(tryptophan),精氨酸(arginine),酪氨酸(tyrosine),组氨酸(histidine), 谷氨酸(glutamic acid),赖氨酸(lysine),苏氨酸(threonine),甘氨酸(glycine),硝酸钾(KNO3),硝酸钙(Ca(NO3)2),硝酸钠(NaNO3),硝酸镁(Mg(NO3)2),硝酸铜(Cu(NO3)2),硝酸锌(Zn(NO3)2),硝酸铁(Fe(NO3)3),双氧水(H2O2)和葡萄糖的水溶液,分别将滤纸浸入到每种溶液中。晾干之后,在滤纸上滴加一定量的2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯溶液,在365nm UV灯下,观察其颜色变化。结果表明在加入2-(4-(2,4-二硝基苯基磺酰氧基)-3-甲酰基苯基)-4-甲基噻唑-5-羧酸乙酯后,滤纸上均显示荧光,但被半胱氨酸(Cys)、高半胱氨酸(Hcy)、谷胱甘肽(GSH)三种溶液浸过的滤纸显示较强荧光,能够与其他分析物的滤纸进行区分。见图8所示。
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