CN110343099B - 一种基于双光子荧光团的有机化合物及应用 - Google Patents
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Abstract
本发明涉及荧光探针，具体的说是一种基于双光子荧光团的有机化合物及其应用。基于双光子荧光团的有机化合物，结构式Ⅰ所示，并以所述双光子荧光团化合物作为检测TrxR的荧光探针。本发明所述荧光探针，在TrxR存在下，对应的荧光发射强度发生明显变化，可用于TrxR的检测。所述化合物灵敏度高、选择性好。这类化合物作为荧光探针可用于活细胞及小鼠中TrxR水平的检测，对研究TrxR的细胞信号转导，以及进一步研究细胞中生物活性物质的代谢及作用，发现TrxR在健康和疾病中所起的作用，具有重要的生物学意义。
Description
技术领域
本发明涉及荧光探针，具体的说是一种基于双光子荧光团的有机化合物及其应用。
背景技术
硫氧还蛋白还原酶(thioredoxin reductase,TrxR)是一种NADPH依赖的包含FAD结构域的二聚体硒酶,属于吡啶核苷酸-二硫化物氧化还原酶家族成员,分为TrxR1、TrxR2、TrxR3三种。其主要功能是催化NADPH将小分子蛋白之硫氧还蛋白(thioredoxin,Trx)上的-S2还原成(-SH)2的反应,即维持Trx的还原型,而Trx在氧化还原调节和抗氧化防御中有着重要作用。硫氧还蛋白系统还具有多种细胞活性：细胞周期的调控、细胞凋亡过程的控制以及炎症调节。研究认为,TrxR在肿瘤细胞中表达量是正常组织的10倍。因此，TrxR已经成为肿瘤诊断的生物标志物。因此,实现快速、灵敏的检测TrxR具有十分重要的意义。
目前，比色法可用来测量血浆和匀浆组织中的TrxR浓度，但是此方法往往需要试样预处理，比如说细胞破碎、组织匀浆等，以及需要大型的检测设备，因此不适合用于生物体内原位、实时的检测。荧光成像法支撑的可视化研究，不仅能对分析对象进行高时空分辨检测的同时，还不会对分析对象造成侵入式的破坏，因此在细胞水平上可实现原位、实时、动态的可视化检测。因此，该方法已经成为分析生命体内TrxR的重要的检测手段之一。
以前设计用于TrxR成像的荧光探针几乎基于TrxR介导的二硫键切割。尽管这些非共价标记的荧光探针可以到达蛋白质的活性位点并被TrxR还原，但是释放的荧光团分散于蛋白质结合域的周围。因此这些反应探针不能提供关于TrxR的原位信息，这是由于释放的荧光团在高度稀释的条件下可能非特异性地转移出细胞，导致成像分辨率差，因此对比度低。此外，共价标记荧光探针可以特异性与TrxR反应，减少扩散，大大提高了成像分析。然而，这些共价标记的荧光探针可以在蛋白质活性位点上结合，并且需要去除未结合的荧光探针以产生对比，这显着降低了信噪比。为了避免上述问题，需要具有高信噪比的可激活探针。该激活探针在未激活状态下是无荧光或低荧光，在激活状态下变成强荧光。当然，为了实现体内成像，荧光探针应该具有在650-900nm的近红外区域(NIR)中的发射波长范围。由于近红外区域的生物体具有低吸收和低自体荧光，近红外成像可以将背景干扰降至最低，并改善组织穿透性。因此，开发具有良好选择性，可在近红外区进行检测生物体系中TrxR的荧光探针具有重要意义。
发明内容
本发明的目的在于提供一种基于双光子荧光团的有机化合物及应用。
为实现上述目的，本发明采用的技术方案为：
一种基于双光子荧光团的有机化合物，基于双光子荧光团的有机化合物，结构式Ⅰ所示，
一种基于双光子荧光团的有机化合物的应用，所述基于双光子荧光团的有机化合物在定性地检测TrxR中的应用。
所述基于双光子荧光团的有机化合物在定性地检测模拟生理环境下、细胞或生物体内外的TrxR中的应用。
本发明的有益效果：
本发明化合物作为检测TrxR的荧光探针，其在TrxR存在下对应的荧光强度和发射波长发生变化，进而可用于水体系、模拟生理环境和细胞内TrxR的检测，并可大大降低外部检测条件的干扰，提高检测精度。本发明化合物用作荧光探针，可用于细胞内TrxR的检测，这对深入研究TrxR的细胞信号转导，以及进一步研究细胞中生物活性物质的代谢及作用，发现TrxR在健康和疾病中所起的作用，具有重要的生物医学意义。
附图说明
图1为本发明实施例提供的所采用的荧光探针和TrxR不同反应时间的荧光强度变化曲线。
图2为本发明实施例提供的所采用的荧光探针对TrxR的选择性示意图；其中，横坐标从左至右依次为空白、还原型烟酰胺腺嘌呤二核苷酸磷酸、TrxR、谷胱甘肽、半胱氨酸、二硫苏糖醇、N-乙酰半胱氨酸、谷胱甘肽还原酶、硫辛酰胺脱氢酶、牛血清白蛋白、谷胱甘肽过氧化物酶1。
图3为本发明实施例提供的细胞(A549)共聚焦显微镜成像。
图4为本发明实施例提供的采用荧光探针用于检测细胞内TrxR的共聚焦显微镜成像。
图5为本发明实施例提供的采用荧光探针用于荷瘤鼠中肿瘤中的TrxR成像。
具体实施方式
下面结合附图对本发明作进一步的限定，但本发明不限于实施例。
本发明基于双光子荧光团的有机化合物如结构式Ⅰ所示，并以所述双光子荧光团化合物作为检测TrxR的荧光探针。本发明所述荧光探针，在TrxR存在下，对应的荧光发射强度发生明显变化，可用于TrxR的检测。所述化合物灵敏度高、选择性好。本发明化合物作为荧光探针可用于活细胞及小鼠中TrxR水平的检测，对研究TrxR的细胞信号转导，以及进一步研究细胞中生物活性物质的代谢及作用，发现TrxR在健康和疾病中所起的作用，具有重要的生物学意义。
具体，基于双光子荧光团的有机化合物结构式Ⅰ为：
将式Ⅰ化合物加入到待测定水体、模拟生理环境或生物体内，与TrxR反应从而导致荧光由关到开，所得化合物II的结构；
实施例1.基于双光子荧光团的有机化合物的制备：
本发明基于双光子荧光团的有机化合物，在荧光团指定的位置修饰上TrxR的检测基团，得到的相应的双光子荧光团化合物。具体实施例如下：
(1)化合物一的制备
由化合物III(B.Liang,et.al,ACS Chemical Biology，2016,11,425-434.)。将无水K2CO3(1.10g，8.0mmol)和6-溴己醇(1.44g，8.0mmol)加入到1-(4-羟基-3-甲氧基苯基)-7-(5-甲基呋喃-2-基)庚-1,6-二烯-3,5-二酮(0.33g，1mmol)的丙酮溶液中，并在室温搅拌过夜。反应完毕后过滤除去K2CO3，然后减压蒸发。粗产品用柱层析色谱进行纯化，洗脱剂选择EtOAc/CH3OH。得到0.32g黄色固体化合物一，产率为75％。
(2)式I的制备
化合物IV(0.33g，1.0mmol)，N，N'-二环己基碳化二亚胺(DCC，0.220g，1.07mmol)和1-羟基苯并三唑(HOBt，0.145g，1.07mmol)溶于无水四氢呋喃中(50mL)。在氮气保护下，将反应混合物在室温下搅拌30分钟。然后将化合物III(0.852g，2.0mmol)加入到混合物中并搅拌12小时。蒸发溶剂，通过柱色谱(EtOAc/CH3OH)纯化粗产物，得到0.32g黄色油状的式I，产率为43％。
所述化合物IV(G.Masanta,et.al,Chemical communication，2012,48,3518-3520.)。
实施例2
将制备所得式Ⅰ化合物作为探针应用于水体系、模拟生理环境和细胞内进行对TrxR的检测，以下模拟生理条件的各项实验均在pH 7.4条件下进行(Tris-HCl缓冲溶液，浓度为10mM)，探针浓度采用10μM。以下细胞内各项实验，探针浓度采用1μM。
上述制备所得式Ⅰ化合物作为探针对TrxR的响应：
加入1mL TrxR(1μM)到10mL的比色管，然后，用10mM Tris-HCl缓冲液(pH 7.4)稀释到10.0mL。最后加入式Ⅰ化合物(10μM)。将各个比色管中工作液分别吸取1m L倒入到荧光皿中测定荧光光谱，在430-680nm处测定荧光强度(参见图1)。如图1所示。上述式Ⅰ化合物可用于实现生物体内的TrxR检测，探针与TrxR反应后得到化合物II，结构如下：
实施例3
式Ⅰ化合物对TrxR的选择性
取多个10mL比色管，并分别在每个10mL比色管中加入1mL待测物，然后用Tris-HC缓冲液(10mM，pH为7.4)定容到10ml，最后加入式Ⅰ化合物(10μM)。将混合液平衡10分钟后，将各个比色管中工作液分别吸取1m L倒入到荧光皿中测定荧光强度。在555nm处测定荧光强度(参见图2)，待测物依次为：1、空白；2、还原型烟酰胺腺嘌呤二核苷酸磷酸(200μM)；3、TrxR(200nM)；4、谷胱甘肽(1mM)；5、半胱氨酸(1mM)；6、二硫苏糖醇(1mM)；7、N乙酰半胱氨酸(1mM)；8、谷胱甘肽还原酶(20μg/mL)；9、硫辛酰胺脱氢酶(20μg/mL)；10、牛血清白蛋白(500μg/mL)；11、谷胱甘肽过氧化物酶1(200μg/mL)。
实施例4
式Ⅰ化合物用于细胞内TrxR的检测
小鼠肺癌细胞A549按照American type Tissue Culture Collection规定进行培养至细胞密度为50％左右。首先，细胞置于共聚焦荧光显微镜下于635nM激发光下拍照。细胞没有荧光生成(图3)。在37℃下，细胞用式Ⅰ化合物(1μM)培养15分钟，用PBS洗涤三次，置于共聚焦荧光显微镜下于635nM激发光下拍照(参见图4)。如图4所示，有明显的荧光生成。因此，探针可以用于检测细胞内源的TrxR。
实施例5
式Ⅰ化合物用于荷瘤鼠肿瘤内TrxR的检测
肺癌小鼠的饲养及实验按照按照“国家实验动物关爱和使用实验指南”的指导原则进行，实验方案经滨州医学院动物管理与使用委员会批准。将式Ⅰ化合物(1μM)注射到小鼠中。随后，整个肿瘤处发射荧光(图5)。因此，探针可以用于检测荷瘤鼠中肿瘤处TrxR。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。作为荧光染料是本发明新化合物的一种用途，不能认定本发明的化合物仅用于荧光染料，对于本发明所属技术领域的普通技术人员来说，在基于本发明化合物用作荧光染料的相同作用机理的考虑下，还可以做出若干简单推理，得出本发明的化合物的其他应用用途，都应当视为属于本发明的保护范围。
Claims (3)
1.一种基于双光子荧光团的有机化合物，其特征在于：基于双光子荧光团的有机化合物，结构式Ⅰ所示，
式Ⅰ。
2.一种权利要求1所述的基于双光子荧光团的有机化合物的应用，其特征在于：所述基于双光子荧光团的有机化合物在制备定性地检测TrxR探针中的应用。
3.按权利要求2所述的基于双光子荧光团的有机化合物的应用，其特征在于：所述基于双光子荧光团的有机化合物在制备定性地检测模拟生理环境下、细胞或生物体内外的TrxR探针中的应用。
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