CN107383099B - 一种基于花菁的有机化合物及其应用 - Google Patents
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Abstract
本发明涉及检测超氧阴离子自由基(O2 ·‑)的近红外荧光探针，具体地说一种基于花菁的有机化合物及其应用。化合物如结构式Ⅰ所示，以所述化合物作为O2 ·‑‑的荧光探针。本发明O2 ·‑荧光探针的这类化合物，在O2 ·‑存在下对应的荧光波长发生明显位移，可用于O2 ·‑的检测，并可大大降低外部检测条件的干扰，提高检测精度。此发明O2 ·‑荧光探针的这类化合物，在O2 ·‑存在时，紫外吸收亦发生明显变化，可同时用紫外分光光度计及肉眼进行检测。这类化合物作为荧光探针可用于细胞内外O2 ·‑水平的检测，这对深入研究O2 ·‑在生物体内的产生、输送及累积等过程的动力学机理，尤其是研究O2 ·‑在线粒体中的生理作用具有重要的生物医学意义。
Description
技术领域
本发明涉及检测超氧阴离子自由基(O2 ·-)的近红外荧光探针，具体地说一种基于花菁的有机化合物及其应用。
背景技术
超氧阴离子自由基(O2 ·-)是细胞中一种重要活性氧自由基，主要产生在线粒体。O2 ·-是一种涉及多种生理和病理过程(包括先天免疫和代谢稳态)的重要细胞信号分子。生物体内O2 ·-的含量变化及代谢失调均会导致一些疾病的发生。异常生成O2 ·-可能导致生物分子的氧化损伤。它的二次产物，如过氧化氢，羟基自由基，过氧化亚硝酰根离子和次氯酸酸也参与信号转导和多种病理过程，包括肺部动脉高血压，心肌病，动脉粥样硬化，缺血再灌注损伤，糖尿病，类风湿关节炎，自闭症，阿尔茨海默病，帕金森病，肌萎缩性侧索硬化和癌症。通过测定生物体内O2 ·-的含量,可实现对某些代谢疾病的诊断。因此,实现快速、灵敏的检测O2 ·-具有十分重要的意义。
目前，用于检测O2 ·-的方法包括:电子顺磁共振法，高效液相色谱法、化学发光法、荧光分析法以及电化学方法等。在上述方法中，荧光法相比较而言更加具有吸引力，不仅简单易行，便于操作，具有高灵敏度、高选择性的特点，而且可以实现活细胞内O2 ·-的“原位可视化”检测，从而对其在生命体内进行“实时在线”观测。Dan Yang等公开了一类用以检测O2 ·-的荧光探针(D.Yang et.al,J.Am.Chem.Soc.,2015,137,6837-43129)，与O2 ·-作用后荧光增强从而检测O2 ·-的存在。但是这类荧光探针对O2 ·-响应慢，检出限高，不能用于快速地检测O2 ·-。而且此探针的激发发射波长位于紫外区，不能有效避免生物自体荧光的干扰，同时紫外光对生物体光漂白作用很大，易于损伤生物样品。为达到充分穿透到组织内部和避免细胞自发荧光干扰目的，大大降低外部环境的干扰，实现定量检测，仍然需要开发具有较长激发发射波长的可操作性荧光探针。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O2 ·-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花菁的有机化合物及其应用。
为实现上述目的，本发明采用的技术方案为：
一种基于花菁的有机化合物，基于花菁的有机化合物结构式如式Ⅰ所示，
一种基于花菁的有机化合物，基于花菁的有机化合物结构式如式II所示，
一种基于花菁的有机化合物的制备方法，
在氮气保护下,(4-溴丁基)三苯基溴化磷和叠氮钠溶于DMF中，于90-100℃条件下，搅拌至溶液由无色到浅黄色到红色，而后冷却至室温，再加入二氯甲烷，直到产生大量的沉淀，过滤收集滤液经萃取收集有机相、旋蒸所得沉淀经二氯甲烷溶解后加入乙酸乙酯得晶体化合物一；
在氮气保护下，将抗坏血酸钠和CuSO4·5H2O的水溶液混合，得到含有铜(I)催化物质溶液，将商业花菁染料、化合物一和DIPEA加入到甲醇中，而后滴加至上述含有铜(I)催化物质溶液中，在氮气保护下，在室温下反应、反应物经纯化即为式II所示化合物；
在氮气保护下，将式II所示化合物溶解在吡啶和二氯甲烷的混合物中，加入三氟甲磺酸酐，将所得混合物在-78--82℃下搅拌20-30分钟，然后在室温下再搅拌30-40分钟，然后，用10mL饱和NaHCO3水溶液淬灭反应，加入乙酸乙酯，并用盐酸中和，而后用盐水洗涤、纯化即得式Ⅰ所示化合物。
一种基于花菁的有机化合物的应用，所述式Ⅰ所示的基于花菁的有机化合物在定量/定性检测超氧阴离子自由基(O2 ·-)中的应用。
所述式Ⅰ所示的基于花菁的有机化合物用于定性/定量的检测生理环境下、细胞或生物体内外的O2 ·-超氧阴离子自由基(O2 ·-)中的应用。
所述式Ⅰ所示的基于花菁的有机化合物用于定性/定量的检测线粒体中的O2 ·-超氧阴离子自由基(O2 ·-)中的应用。
一种检测超氧阴离子自由基(O2 ·-)的近红外荧光探针，所述探针为式Ⅰ所示花菁的有机化合物，
所述式Ⅰ所示的基于花菁的有机化合物用于定性/定量的检测生理环境下、细胞或生物体内外的O2 -超氧阴离子自由基(O2 -)中的应用。
所述式Ⅰ所示的基于花菁的有机化合物与生理环境下、细胞或生物体内外的O2 -超氧阴离子自由基(O2 -)反应形成式II所示化合物，进而实现对O2 -超氧阴离子自由基(O2 -)的定性/定量的检测。
本发明的有益效果：
本发明用于作为O2 ·-荧光探针的化合物，其在O2 ·-存在下对应的荧光强度和发射波长发生变化，同时紫外吸收也对应发生改变，进而可用于水体系、模拟生理环境和细胞内O2 ·-水平的检测，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O2 ·-的检测，而且还可对细胞内的线粒体进行定位，这对深入研究O2 ·-在生物体内的产生、输送及累积等过程的动力学机理，进一步了解O2 ·-的生理作用，尤其是研究O2 -在线粒体对抗氧化应激环境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采用的荧光探针对O2 ·-检测前后紫外吸收变化。
图2为本发明实施例提供的采用的荧光探针对O2 ·-检测前后荧光变化。
图3为本发明实施例提供的所采用的荧光探针与O2 ·-作用后在742nm和790nm处荧光强度比率值变化曲线图。
图4为本发明实施例提供的所采用的荧光探针对O2 -的选择性示意图；其中，横坐标从左至右依次为：O2 ·-、空白对照、羟基自由基、单线态氧、次氯酸、过氧化氢、脂质过氧化物、亚硝酰氢、过氧化亚硝酰阴离子、亚硝酸根离子、一氧化氮、过氧化叔丁醇、过氧化甲基亚油酸盐和枯烯过氧化氢。
图5为本发明实施例提供的采用荧光探针用于检测细胞线粒体内O2 -的共聚焦显微镜成像。
具体实施方式
下面结合附图及实施例用于进一步说明本发明，但本发明不限于实施例。
本发明化合物如结构式Ⅰ所示，以所述化合物作为O2 ·-的荧光探针。本发明O2 ·-荧光探针的这类化合物，在O2 ·-存在下对应的荧光波长发生明显位移，可用于O2 ·-的检测，并可大大降低外部检测条件的干扰，提高检测精度。此发明O2 ·-荧光探针的这类化合物，在O2 ·-存在时，紫外吸收亦发生明显变化，可同时用紫外分光光度计及肉眼进行检测。这类化合物作为荧光探针可用于细胞内外O2 ·-水平的检测，这对深入研究O2 ·-在生物体内的产生、输送及累积等过程的动力学机理，尤其是研究O2 ·-在线粒体中的生理作用具有重要的生物医学意义。
实施例1
基于花菁的有机化合物结构式为：
式Ⅰ化合物与待测定水体、模拟生理环境或生物体内外中的O2 ·-结合，得结构式II结构的化合物从而导致式Ⅰ化合物的荧光强度和波长的改变，以及紫外吸收的改变，进而利用式Ⅰ化合物在O2 ·-下形成式II化合物可对O2 ·-进行定性、定量的检测。
基于花菁的式Ⅰ有机化合物的制备：
(1)化合物一的制备
在氮气保护下,(4-溴丁基)三苯基溴化磷(14.35g,30mmol)和叠氮钠(3.9g,60mmol)溶于50mL DMF中。90℃条件下，搅拌，过夜。溶液的颜色由无色到浅黄色到红色。将反应瓶冷却到室温，加入50mL二氯甲烷，直到产生大量的沉淀。过滤，收集滤液。用50mL二氯甲烷和50mL蒸馏水萃取三次，收集有机相，旋蒸。旋蒸后的产物置入圆底烧瓶中，安装回流装置，加热10min，搅拌，加入约7.5毫升的二氯甲烷使其完全溶解，40℃回流30min。溶液微沸时，加入乙酸乙酯，使溶液有白色晶体析出又消失，直至浑浊出现时，再加入二氯甲烷，如此重复3次，冷却，析出晶体，得到化合物一。
1H NMR(400MHz,CDCl3)δ(ppm):7.89-7.85(q,6H),7.82-7.79(t,3H),7.73-7.71(m,6H),3.98-3.92(m,2H),3.46-3.44(t,2H),2.06-2.01(m,2H),1.76-1.74(m,2H).13C NMR(100MHz,CDCl3,ppm)δ134.98,133.72,133.64,130.52,130.42,118.54,117.86,50.59,29.19,29.06,22.34,21.93,19.84,19.80.GC-MS(API-ES):m/z C22H23N3P+[M]+Calcd:360.1624,found:360.4022.
(2)化合物二的制备
在氮气保护下，将抗坏血酸钠(0.01M，1mL)和CuSO4·5H2O(0.01M，1mL)的水溶液混合，得到含有铜(I)催化物质溶液。将商业花菁染料(63.6mg,0.1mmol)，化合物一(39.6mg，0.11mmol)和DIPEA(1.5g，0.01mmol)加入到6.0mL甲醇中，通过恒压滴液漏斗滴加到上述含有铜(I)催化物质溶液中。将混合物在氮气保护下，在25℃下进一步搅拌24小时。真空除去溶剂，所得蓝色固体残余物用梯度洗脱剂CH2Cl2和CH3OH(100:0-85:15，v/v)的硅胶色谱纯化(200-300目)，即得到式II所示化合物。
1H NMR(400MHz,CD3OD)δ(ppm):8.07(s,2H),7.90-7.88(m,6H),7.38-7.37(d,3H),7.32-7.29(m,6H),7.10-7.08(t,3H),7.04-7.00(t,5H),5.75-5.72(d,2H),5.25(s,5H),5.12(s,3H),4.55-4.52(t,3H),4.14-4.08(m,1H),3.43-3.37(m,1H),3.10-3.08(m,2H),2.97-2.93(m,3H),2.69-2.64(m,3H).2.20-2.18(m,3H),2.03(s,1H),1.85-1.81(m,1H),1.76-1.75(d,2H),1.50-1.46(m,1H),1.29-1.21(m,12H),1.05-1.00(m,2H),0.94-0.92(m,2H).13C NMR(100MHz,CD3OD)δ(ppm):169.97,166.81,159.96，157.15,143.82,140.03,137.84,134.94,133.49,133.41,130.26,130.16,128.00,127.91，124.09,122.39,121.69,118.63,117.94,114.55,108.27,93.21,93.08,62.27,61.06,52.30,49.95，38.33,36.31,33.07,30.30,30.17,29.24,29.11,27.70,27.56,21.08，20.93,20.66,20.52,19.49,19.46,19.09,10.27,7.87,6.34.GC-MS(API-ES):m/z C66H73N6OP+[M]+Calcd:996.557,found:498.6522.
(3)化合物三的制备
在氮气保护下，将式II所示化合物(100mg，0.1mmol)溶解在吡啶(5mL)和二氯甲烷(5mL)的混合物中，加入三氟甲磺酸酐(0.11mmol)，将所得混合物在-78℃下搅拌20分钟，然后在室温下再搅拌30分钟。然后，用10mL饱和NaHCO3水溶液淬灭反应。加入乙酸乙酯(50mL)，并用盐酸(150mL，1M)中和，而后用盐水洗涤。有机层用无水硫酸镁干燥，真空浓缩即得到式Ⅰ绿色粘稠固体，而后经梯度洗脱剂CH2Cl2和CH3OH(100:0-85:15，v/v)的硅胶色谱纯化(200-300目)。
1H NMR(400MHz,CD3OD)δ(ppm):8.77(s,1H),7.87-7.79(m,16H),7.72-7.70(m,9H),7.40-7.37(m,1H),7.13-7.12(m,1H),7.07-7.01(m,3H),6.79-6.77(d,1H),5.48-5.23(m,4H),4.93-4.92(m,3H),4.13-4.04(m,5H),3.81-3.78(m,3H),3.60(s,2H),2.90-2.87(m,3H),2.53-2.41(m,9H),1.43-1.41(m,2H).1.27-1.26(m,9H).13C NMR(100MHz,CD3OD,ppm)δ167.62,166.81,158.46，156.25,141.42,141.03,139.84,136.54,132.99,131.45,130.99,130.16,128.60,127.91，125.59,123.49,122.39,119.63,118.55,115.55,109.37,93.51,93.28,63.27,62.06,53.33,49.93，38.03,35.33,33.27,30.38,30.19,29.29,29.21,27.80,27.86,22.48，22.43,21.56,21.55,19.59,19.36,19.19,10.17,7.89,6.55.GC-MS(API-ES):m/z C67H72F3N6O3PS+[M]+Calcd:1128.5071,found:564.8912.
实施例2
将制备所得式Ⅰ所示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O2 ·-的检测，模拟生理条件，以下各项实验均在pH＝7.4条件下进行(HEPES缓冲溶液，浓度为40mM)，探针浓度采用10μM。
上述制备所得式Ⅰ所示化合物对O2 ·-的紫外响应：
pH采用HEPES缓冲溶液控制。于每个10mL比色管中加入10μM式Ⅰ化合物，再加入不同pH的40mM HEPES，然后加入10μM O2 ·-，超纯水定容到10ml，摇匀溶液，平衡10min后，将上述工作液加入比色皿中测定紫外吸收光谱。紫外吸收光谱在检测O2 ·-前后的变化如图1所示，式Ⅰ所示化合物可用于实现生物体内的O2 ·-检测。同时，式Ⅰ所示化合物与O2 ·-反应后产物结构如下：
实施例3
式Ⅰ所示化合物对O2 ·-的荧光响应：
pH采用HEPES缓冲溶液控制。于每个10ml比色管中加入10μM化合物式一，再加入不同pH的40mM HEPES，然后加入10μM O2 ·-，超纯水定容到10ml，摇匀溶液，平衡10min后，将上述工作液加入荧光皿中测定荧光光谱。荧光光谱在检测O2 -前后的变化如图2所示。式Ⅰ所示化合物可用于实现生物体内的O2 -检测。
由图2表示随O2 ·-浓度的变化体系荧光强度的变化，表明随O2 -浓度的增加，体系630-830nm波段的荧光强度明显增强,780-810nm波段的荧光强度明显减弱。
实施例4
式Ⅰ所示化合物O2 ·-的定量检测
于10ml比色管中加入10μM式Ⅰ所示化合物，再加入40mM HEPES(pH＝7.4)，然后加入10μM O2 ·-，超纯水定容到10ml，摇匀溶液，平衡10min后，将上述工作液加入荧光皿中测定荧光光谱。
分别取742nm和790nm处荧光强度比率值，输入软件OriginPro 8.0,得到线性工作曲线如图3所示，其中线性拟合曲线的线性回归常数为0.9954，表明本式Ⅰ所示化合物能定量的测定O2 ·-的浓度。
实施例5
式Ⅰ所示化合物对O2 ·-的选择性
取多个10ml比色管，并在每个10ml比色管中加入10μM式Ⅰ所示化合物，再加入40mMpH为7.4的HEPES缓冲液，然后分别20μM O2 ·--以及200μM其他待测物最后用超纯水定容到10ml。摇匀溶液，25℃下平衡10min后，将各个比色管中工作液分别倒入到荧光皿中测定荧光光谱(参见图4)。待测物依次为：O2 ·-、空白对照、羟基自由基、单线态氧、次氯酸、双氧水、脂质过氧化物、亚硝酰氢、过氧化亚硝酰阴离子、亚硝酸根离子、一氧化氮、过氧化叔丁醇、过氧化甲基亚油酸盐和枯烯过氧化氢。由图4可知式Ⅰ所示化合物对O2 ·-具有很好的选择性。
实施例6
式Ⅰ所示化合物用于细胞线粒体内O2 ·-的检测：
小鼠肝癌细胞HepG2细胞按照American type Tissue Culture Collection规定进行培养。10.0μM式Ⅰ所示化合物孵育HepG2细胞10分钟，用培养基洗涤3次，置于共聚焦荧光显微镜下拍照，结果如图5a所示；然后加入1μM rhodamine 123(商品化线粒体染色染料)孵育HepG2细胞10分钟，用培养基洗涤3次，置于共聚焦荧光显微镜下拍照,结果如图5b所示；然后再加入1μM Hoechest(商品化细胞核染色染料)孵育HepG2细胞10分钟，用培养基洗涤3次，置于共聚焦荧光显微镜下拍照，结果如图5c所示；
由图5可见其中，图5d是图5a,图5b和图5c叠加图，图5a和图5b重合，图5a和图5c没有重合。表明式Ⅰ所示化合物主要对线粒体染色。
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 (5)
1.一种基于花菁的荧光探针，其特征在于：基于花菁的荧光探针阳离子结构式如式Ⅰ所示，
式Ⅰ。
2.一种基于花菁的有机化合物，其特征在于：基于花菁的有机化合物阳离子结构式如式II所示，
式II。
3.一种权利要求1所述的基于花菁的荧光探针的应用，其特征在于：所述基于花菁的荧光探针在非疾病诊断中定量/定性检测超氧阴离子自由基中的应用。
4.按权利要求3所述的基于花菁的荧光探针的应用，其特征在于：所述基于花菁的荧光探针在非疾病诊断中定性/定量的检测生物体内外的O2 ·-超氧阴离子自由基中的应用。
5.按权利要求4所述的基于花菁的荧光探针的应用，其特征在于：所述基于花菁的荧光探针在非疾病诊断中定性/定量的检测线粒体中的O2 ·-超氧阴离子自由基中的应用。
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