第46卷第7期2018年4月广　州　化　工Guangzhou Chemical IndustryVol.46No.7Apr.2018谷胱甘肽荧光探针的研究进展*石　磊1,2,黄　玲3,龚盛昭1,2(1广东轻工职业技术学院轻化工技术学院,广东　广州　510300;2广东省绿色日用化工工程技术研究中心,广东　广州　510300;3佛山市安安美容保健品有限公司,广东　佛山　528099)摘　要:谷胱甘肽在生物体的许多生理过程中发挥着重要作用,所以细胞内谷胱甘肽含量的检测对细胞功能研究和病理分析都具有重要的意义㊂以荧光探针为基础的荧光分析法因其操作简便㊁灵敏度高和专一性强等优点而备受大家关注,并且有机小分子荧光探针还可以应用于活体细胞和生物体的成像技术㊂本文主要综述了近年来谷胱甘肽荧光探针的研究现状,并按照谷胱甘肽与探针识别基团的识别机理分类阐述,同时对谷胱甘肽荧光探针的未来发展趋势进行了展望㊂关键词:谷胱甘肽㊁荧光探针㊁识别机理㊁检测　中图分类号:O657.3　文献标志码:A文章编号:1001-9677(2018)07-0023-06*基金项目:广东轻工职业技术学院人才类项目(项目编号:KYRC2017-0031)㊂第一作者:石磊(1985-),男,博士,讲师,主要从事荧光探针的合成与应用㊂通讯作者:龚盛昭㊂Research Progress on Fluorescent Probes for Glutathione *SHI Lei 1,2,HUANG Ling 3,GONG Sheng -zhao 1,2(1School of Chemical Engineering and Technology,Guangdong Industry Polytechnic,Guangdong Guangzhou 510300;2Guangdong Engineering Technical Research Center for Green Household Chemicals,Guangdong Guangzhou 510300;3Foshan Anan beauty &Health products Co,Ltd,Guangdong Foshan 528099,China)Abstract :Glutathione plays an important role in many physiological processes of life system,and the detection of glutathione in cell is significant for the research of cell function and pathological analysis.Fluorometric analysis based on fluorescent probes has attracted much attention due to its advantages,such as simple operation,high sensitivity and specificity.Moreover,the organic fluorescent probes could also be applied to bioimaging technology for living cells and organisms.The research progress on glutathione fluorescent probes was introduced and classified according to the recognition mechanism between glutathione and recognition groups of probes,and the developing trends of fluorescent probes for glutathione were prospected.Key words :glutathione;fluorescence probe;recognition mechanism;detection谷胱甘肽(Glutathione,缩写GSH)是一种含有巯基㊁氨基和γ-酰胺键的三肽,主要由谷氨酸㊁半胱氨酸和甘氨酸组成㊂谷胱甘肽是细胞内一种重要的调节代谢物质;它不仅能够清除体内的过氧化物及其他自由基,促进肝脏酶活性㊁解毒和维持红细胞膜完整性等作用,同时还具有维持DNA 的生物合成和细胞免疫等多种生理功能[1-2]因此,检测生物体中的GSH 含量对于一些疾病的预防㊁研究和治疗都具有十分重要的作用,故而引起了诸多科研工作者的高度关注[3-4]㊂相比于分光光度法㊁色谱法㊁毛细管电泳法㊁电化学法等传统检测方法,以荧光探针为基础的荧光分析法具有测试简单㊁选择性高㊁响应时间短等优点㊂更重要的是,荧光探针还能应用于生物体内的实时监测和生物成像研究,故而被广泛应用于生物医学㊁分析化学和化学生物学等诸多领域[5-6]㊂近年来,基于谷胱甘肽的荧光探针得到了迅猛发展;若按照谷胱甘肽与荧光探针识别基团的识别机理进行分类,可以将其分为加成反应取代反应和还原反应㊂本文主要综述了近年来谷胱甘肽荧光探针的设计合成与应用进展,并分类阐述如下㊂1　加成反应加成反应是利用GSH 中具有亲核性的巯基与不饱和双键(主要是碳碳双键)发生加成反应,使得探针的荧光发射光谱发生变化,从而实现对检测对象的识别与检测㊂1.1　马来酰亚胺类自Kanaoka [7]首次报道了以马来酰亚胺作为生物硫醇识别基团的荧光探针以来,基于马来酰亚胺的香豆素㊁BODIPY㊁喹啉㊁萘酐等[8-10]荧光探针陆续涌现出来,并成功应用于生物体内GSH 的选择性识别(图1)㊂然而,按此原理构建的大部分荧光探针对半胱氨酸(Cys)㊁同型半胱氨酸(Hcy)和GSH 均有响应,很难对这三者进行区分;仅少许报道是例外㊂其中,24　广　州　化　工2018年4月Gunnlaugsson 等[11]合成了一类含有马来酰亚胺的稀土荧光材料5;该荧光材料对于GSH 具有很强的专一性识别能力,而对其它氨基酸则表现出差异性的荧光响应,从而能够很好区分Cys 和Hcy 等其它氨基酸㊂图1　基于马来酰亚胺类识别基团的荧光探针1~5Fig.1　Fluorescent probe 1~5based on the maleimide recognition group1.2　α,β-不饱和双键和马来酰亚胺类似,α,β-不饱和丙二腈基团㊁α,β-不饱和酮和芳基乙烯基也可以作为小分子硫醇的迈克尔加成反应位点㊂基于此项设计,Kwon 等[12]㊁Chen 等[13]和Chang 等[14]合成了三种荧光探针6㊁7和8(图2),并实现了在细胞体内Cys㊁Hcy 和GSH 等生物硫醇的识别和检测㊂然而遗憾的是,这三种荧光探针无法对生物硫醇进行很好区分㊂图2　基于α,β-不饱和双键识别基团的荧光探针6~8Fig.2　Fluorescent probe 6~8based on the recognition group ofα,β-unsaturated double bond1.3　硝基乙烯图3　基于硝基乙烯识别基团的荧光探针9和10Fig.3　Fluorescent probe 9,10based on the nitroethylenerecognition group2014年,Akkayatwi 等[15]报道了一种利用硝基乙烯基为识别基团的BODIPY 探针9(图3)㊂该荧光探针与GSH 生成硫醚化合物后,抑制了PET 效应,从而使荧光强度大大增强㊂基于PET 和ICT 效应,Zhou 等[16]同样报道了基于硝基乙烯的咔唑荧光探针10,并且实现了从Cys 和Hcy 等氨基酸中高选择性识别出GSH㊂1.4　丙烯酸酯类从2011年开始,基于丙烯酸酯识别基团的多种荧光探针不断被报道出来[4,17-18],并成功应用于生物硫醇的小分子检测㊂相比于GSH 与丙烯酸酯所生成的反应产物,Cys 和Hcy 结构中的氨基可以进一步发生环化反应而使丙烯酸酯离去,所以基于这类识别机理的大部分荧光探针都是用于选择性识别Cys 和Hcy㊂然而,Wang 等[19]所设计了1,3,5-三芳基吡唑啉荧光探针11则有所不同(图4)㊂该荧光探针和GSH 所生产的加成产物具有较高量子产率,而Cys 和Hcy 等巯基氨基酸则与丙烯酸酯形成环化离去,生成具有较低量子产率的酚类化合物,从而实现了对GSH 的高选择性识别和检测㊂图4　基于丙烯酸酯识别基团的荧光探针11Fig.4　Fluorescent probe 11based on the recognition group of acrylate2　取代反应GSH 中的巯基不仅可以发生加成反应,也能够发生亲核取代反应,从而使探针分子结构中某些基团被离去㊂基于该类反应机制的GSH 识别基团主要有:卤素类㊁芳基醚类㊁磺酸酯/磺酰胺类㊁硫醋类等㊂2.1　卤素基团利用Cys /Hcy 结构中氨基与巯基距离较近的结构特点和产物性能的差异,Chen 等[20]和Lin 等[21]设计了2种选择性识别GSH 的BODIPY 荧光探针12和香豆素荧光探针13,并实现了细胞体内GSH 的实时检测与生物成像(图5)㊂2014年,Guo 等[22]报道了一种含3个反应位点的荧光探针14㊂由于GSH 中氨基和巯基相距较远,GSH 中巯基将先与探针先发生亲核取代反应,继而氨基与苯并噻唑中的3号反应位点发生加成反应㊂正是三种生物硫醇的结构特征和产物的不同,使得该荧光探针能够利用不同的荧光通道实现Cys㊁Hcy 和GSH 的高选择性检测㊂同样基于含3个反应位点的香豆素探针,Han 等[23]报道了另一种香豆素荧光探针15,该探针不仅可以选择性检出GSH,而且对于H 2S 也具有很好的检测和分辨能力㊂第46卷第7期石磊,等:谷胱甘肽荧光探针的研究进展25图5　基于卤素取代反应的荧光探针12~15Fig.5　Fluorescent probe 12~15based on substitution reaction of halogen2.2　芳基醚(氧㊁硫㊁硒醚)图6　基于芳基醚取代反应的荧光探针16和17Fig.6　Fluorescent probe 16,17based on substitution reaction of aryl ether2014年,Wang 等[24]和Lim 等[25]先后报道了含间硝基和对(硝基偶氮基)苯基醚的花菁染料探针16和17(图6)㊂上述两种荧光探针可以与GSH 发生亲核取代反应,使得芳基醚离去,并破坏PET 效应,而且这两种探针对GSH 都具有很好的专一检测能力㊂同样基于Cys /Hcy 取代-重排反应和GSH 取代反应的产物差异,Guo 等[26]报道了含对甲氧基苯硫醚基团的荧光探针18,并实现了激发波长分别为455nm 和588nm 的双通道选择性识别与检测(图7)㊂Yang 等[27]则报道了基于ESIPT 和PET 两种发光机理的荧光探针19;Cys /Hcy 与探针分子先后发生巯基和氨基的取代反应,进而生成内醋,最终产物通过ESIPT 机理发出蓝色荧光;然而,探针19则与GSH 只发生酯基的交换反应,阻断原有的PET 效应,最终释放出罗丹明荧光团的红色荧光㊂图7　基于芳基硫醚取代反应的荧光探针17和18Fig.7　Fluorescent probes 17and 18based on substitution reactionof arylthioether图8　基于双识别基团的荧光探针20~22Fig.8　Fluorescent probe 20~22based on double recognition groups2015年,Zhu 等[28]报道了一种同时具备二硫键和芳基硫醚两个独立反应位点的BODIPY 荧光探针20(图8)㊂虽然生物硫醇都能断裂二硫键,并形成五元环离去,但是Cys /Hcy 由于存在巯基-氨基的转换,故而产生差异性的荧光发射光谱,从而实现对GSH 的选择性检测㊂之后,Chen 等[29]和Mulay 等[30]也发表了基于双识别基团的香豆素荧光探针21和22,并利用2个识别基团的联合作用,实现了从其它巯基氨基酸中选择性识别GSH;此外,探针21对H 2S 也具有很好的选择性识别能力㊂2.3　磺酸酯/磺酰胺类磺酸酯/磺酰胺键在生物硫醇的作用下很容易断裂,从而26　广　州　化　工2018年4月释放出荧光信号和SO 2㊂基于此项设计的多种荧光探针不断被报道出来[31-32]㊂然而遗憾的是,这类荧光探针普遍难以实现生物硫醇的选择性识别(图9)㊂图9　基于磺酸酯/磺酰胺键荧光探针的识别机理图Fig.9　The diagram of fluorescent probes based on recognition groupof sulfonyl ester or sulfonamide bond为提升磺酰胺基团的选择性,Yin 等[33]和Liu 等[34]创新性地将苯基改为萘环和氧化硫代吗啉,设计了花菁染料23和萘酐荧光探针24(图10);这两种荧光探针在对GSH 的选择性识别方面都取得了较大突破㊂图10　基于磺酰胺识别基团的荧光探针23和24Fig.10　Fluorescent probe 23,24based on sulfonamide recognition groups2.4　氮-硒键早在2007年和2009年,唐波课题组[35-36]先后发表了利用N-Se 键断裂来识别GSH 的罗丹明荧光探针25和26(图11),并成功应用于细胞体内GSH 的检测和成像㊂图11　基于氮-硒键识别基团的荧光探针25和26Fig.11　Fluorescent probe 25,26based on the recognition group ofnitrogen-seleniumbonds图12　基于氮-硒键识别基团的花菁探针27和28Fig.12　The cyanine probe 27,28based on the recognition groupof nitrogen-selenium bonds2012年,Chen 等[37]则将N-Se 键识别位点引入到花菁染料中,合成了用于检测GSH 的近红外荧光探针27(图12)㊂同样是基于花菁染料,唐波课题组[38]则将含N-Se 键的五元环引入到荧光探针28结构中㊂在GSH 作用下N-Se 键断裂,由于产物内存在PET 效应导致荧光淬灭;而此时如果加入H 2O 2,则上述苯硒酚又重新被氧化,并伴随794nm 处荧光强度的不断増强㊂2.5　硒-硒键GSH 不仅可以断裂N-Se 键,也可以断裂Se -Se 键㊂Lou等[39]报道了一种以Se-Se 键为GSH 识别位点的荧光素探针29;该荧光探针不仅可以用于GSH 的快速检测,而且还能用于监视细胞体内GSH 与活性氧之间的氧化还原变化(图13)㊂图13　基于硒-硒键识别基团的荧光探针29Fig.13　Fluorescent probe 29based on recognition groupof selenium-selenium bond3　还原反应GSH 结构中的巯基不仅具有亲核性,同时还具有还原性;并且基于硫醇还原性反应的荧光探针也不断被报道出来[40-41]㊂其中,双硫键-氨基甲酸酯和双硫键-苯乙酸酯等基团是用来构建硫醇荧光探针的常见方法,并被引入到氨基萘乙酮[42]㊁卟啉[43]㊁荧光素[44]等荧光团结构中(图14)㊂然而,此类方法对生物硫醇的选择性识别能力却较为有限㊂图14　以还原反应作为识别机理的探针27和28Fig.14　Fluorescent probe 27and 28based on the recognitionmechanism of reductionreactions图15　利用GSH 还原碲进行识别的荧光探针33Fig.15　Fluorescent probe 33based on the reduction of tellurium by GSH第46卷第7期石磊,等:谷胱甘肽荧光探针的研究进展272013年,Han等[45]报道了一种利用GSH还原碲的花菁染料探针33(图15)㊂该探针在GSH等硫醇作用下发生还原反应,导致体系荧光在820nm处大大减弱;不仅如此,该还原产物又能被ONOO-氧化,同时伴随着荧光强度的急剧增强㊂4摇结论与展望从上述的国内外研究进展来看,近年来基于GSH的荧光探针已经被报道了很多,所设计的荧光探针结构和识别基团也变化多样㊂然而,能够在复杂生命体中专一检测谷胱甘肽而完全不受其它硫醇干扰旳荧光探针仍然存在较大的挑战,相关的报道仍在少数㊂此外,随着生命科学与分析技术的不断发展,人们已经开始从亚细胞层去认识和探究生命活动的本质,但基于细胞器靶向的生物硫醇荧光探针研究仍然处于初始阶段㊂因此,未来关于GSH等生物硫醇化合物的荧光探针研究方向可以集中在以下3个方面:(1)设计更多具有专一性检测GSH的识别基团和荧光探针分子;(2)设计与合成出更多具有细胞器㊁组织㊁器官特异性靶向的探针;(3)利用双光子㊁近红外光的生物应用优势,将双光子和近红外荧光探针应用到细胞和动物组织的生物成像中㊂参考文献[1]　TOWNSEND D M,TEW K D,TAPIERO H.The importance ofglutathione in human disease[J].Biomed.Pharmacother,2003,57 (3-4):145-155.[2]　张照明,张海涛,袁利明.国内谷胱甘肽研究进展[J].广州化工,2009,37(3):55-57.[3]　钟华,张慧,许海平.谷胱甘肽的测定方法进展[J].氨基酸和生物资源,2014,36(1):23-26.[4]　谢光杰,户明星,徐丽萍.选择性生物小分子硫醇荧光探针的研究进展[J].化学研究与应用,2016,28(9):1177-1185. 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