Synthesis and Properties of A Mitochondria-targeted Coumarin-based pH Fluorescent Probe

一种线粒体靶向香豆素基pH荧光探针合成及性能 Synthesis and Properties of A Mitochondria-targeted Coumarin-based pH Fluorescent Probe 1 first-author 陈俊(1994-)，男，河南信阳人，硕士研究生，2018年于河南科技学院获得学士学位，主要从事荧光探针的合成及性能的研究。E-mail: 649901225@qq.com http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349174&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349176&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349178&type= 陈俊 (1994-)，男，河南信阳人，硕士研究生，2018年于河南科技学院获得学士学位，主要从事荧光探针的合成及性能的研究。E-mail: 649901225@qq.com 649901225@qq.com 1 1 corresp E-mail: mengxm@ahu.edu.cn E-mail: mengxm@ahu.edu.cn 孟祥明(1980-)，男，安徽六安人，博士，教授，博士研究生导师，2007年于中国科学技术大学获得博士学位，主要从事荧光化学传感器的设计合成及新型医药农药的合成工艺开发的研究。E-mail: mengxm@ahu.edu.cn http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349181&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349184&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349187&type= 孟祥明 (1980-)，男，安徽六安人，博士，教授，博士研究生导师，2007年于中国科学技术大学获得博士学位，主要从事荧光化学传感器的设计合成及新型医药农药的合成工艺开发的研究。E-mail: mengxm@ahu.edu.cn mengxm@ahu.edu.cn 安徽大学 化学化工学院，安徽 　 合肥 　 230601 安徽大学 化学化工学院，安徽 　 合肥 　 230601 School of Chemistry and Chemical Engineering, Anhui University, Hefei 230601, China School of Chemistry and Chemical Engineering, Anhui University, Hefei 230601, China 线粒体作为一种重要的细胞器，其功能包括能量供应、信号传导、细胞分化以及控制细胞生理周期和细胞生长等。线粒体的任何损伤及随后的功能障碍都可能导致一系列人类疾病。其中线粒体pH直接影响着其生化过程，正常的pH水平是线粒体功能正常运作的基础。本文以7-羟基香豆素为原料，合成了一种对线粒体特异性标记的pH荧光探针(CMPH)。并对该探针的光学性质、细胞毒性和线粒体靶向能力进行了研究。结果表明，探针CMPH具有较大的斯托克斯位移，能对pH特异性荧光响应。pH在6.58.2范围内时，探针的荧光强度和pH值具有良好的线性关系。细胞毒性测试表明探针CMPH细胞毒性低。共聚焦荧光成像结果证明，该探针能有效靶向线粒体并能监测线粒体pH的变化。我们的目的是为线粒体pH变化的监测提供一种新的成像工具。 Mitochondrion is an important organelle whose functions include energy supply, signal transduction, cell differentiation, cell death, and the control of cell physiological cycle and cell growth. Any damage to mitochondria and subsequent dysfunction could lead to a range of human diseases. Mitochondrial pH directly affects its biochemical process, and the normal pH level is the basis for the normal operation of mitochondrial function. In this paper, a mitochondria-targeted pH fluorescent probe(CMPH) was synthesized by choosing 7-hydroxycoumarin as the raw material. Its optical properties, cytotoxicity and mitochondria-targeted ability were studied. The results showed that probe CMPH had a large Stokes shift, can specifically give a fluorescent response to pH, and its fluorescent intensities had a good linear relationship to pH values in the range of pH=6.5-8.2. Cytotoxicity testing indicated that probe CMPH had low cytotoxicity. Confocal fluorescence imaging demonstrated that CMPH can efficiently target mitochondria and monitor the change of mitochondrial pH. Our purpose is to provide a new imaging tool for monitoring the change of mitochondrial pH. 荧光探针 香豆素 pH 线粒体 fluorescent probe coumarin pH mitochondria 1　引言 1 线粒体是真核细胞母系遗传的细胞质细胞器，是重要的生物能合成工厂 [ 1 1 ] 。线粒体在细胞新陈代谢、器官、组织以及整个生命体内包括Ca 2+ 平衡的调控 [ 2 2 ] 、清除多余或者破损的细胞器和触发细胞死亡等方面都起着至关重要的作用 [ 3 3 - 4 4 3-4 ] 。因此，细胞的存活需要维持正常的线粒体水平。线粒体缺陷或功能障碍与一些心血管和神经性疾病等有关 [ 5 5 - 7 7 5-7 ] 。而且线粒体的微环境与其功能关系紧密，尤其是pH值直接影响其生化过程。例如线粒体表现出一种略碱性基质，穿过内膜的质子动力能驱动ATP合成、离子及代谢物吸收到基质中去 [ 8 8 ] 。由此可见，线粒体动态变化 [ 9 9 ] 和微环境 [ 10 10 ] 的检测对线粒体生物学及相关疾病的研究具有重要意义。 到目前为止，已报道的pH测定方法包括核磁共振法(NMR) [ 11 11 ] 、吸收光谱法 [ 12 12 - 13 13 12-13 ] 、表面增强拉曼散射法(SERS) [ 14 14 - 15 15 14-15 ] 、水凝胶 [ 16 16 ] 等。而基于pH荧光探针的荧光成像方法因能排除其他信号分子的干扰，对pH具有特异性响应、灵敏度高和实时性等优点而备受关注 [ 17 17 - 18 18 17-18 ] 。开发具有线粒体靶向能力的pH荧光探针将为细胞线粒体内pH波动的实时成像研究提供有力工具。 基于本课题组利用荧光探针可视化研究细胞微环境方面的工作 [ 19 19 - 22 22 19-22 ] ，本文以性能优异的香豆素 [ 23 23 - 25 25 23-25 ] 为荧光团设计并合成了一种靶向线粒体的pH荧光探针(CMPH)。酚羟基是pH响应基团，在pH < 7的酸性条件下，羟基是较弱的电子供体;在pH > 7的碱性条件下，脱质子化的O - 是较强的供电子基团。带正电荷的苯并噻唑盐既赋予探针良好的水溶性 [ 26 26 ] ，又可作为有效的线粒体的靶向基团。通过对光学性能和细胞毒性的测试，表明该探针具有pH特异性和较好的生物相容性。本研究通过对细胞共聚焦荧光成像的分析，评价了探针CMPH用于活细胞线粒体pH特异性荧光成像的可行性，旨在为线粒体微环境的可视化检测及相关疾病的研究提供一种便捷的化学工具。 2　实验 1 2.1　化学试剂 2 7-羟基香豆素(阿拉丁试剂有限公司)、吡啶(天津博迪化工有限股份公司)、三氟乙酸(安耐吉化学)、2-甲基苯并噻唑(安耐吉化学)、乙酸乙酯、二甲基亚砜(DMSO)及其他试剂均为市售分析纯级。 2.2　探针CMPH的合成 2 探针CMPH的合成路线如 图1 图1 所示。 2.2.1　中间体的合成 3 根据文献[ 27 27 ]合成Compound 1(7-乙酰氧基香豆素)和Compound 2(7-羟基香豆素-8-醛)、文献[ 28 28 ]合成Compound 3(3-苄基-2-甲基苯并噻唑溴盐)。 2.2.2　探针CMPH的合成 3 精准地称取一定量的Compound 2(0.53 g,2.8 mmol)和Compound 3(0.897 g,2.8 mmol)加入史莱克瓶中，在氮气保护下加入乙醇(10 mL)和哌啶(3滴),70 ℃下反应8 h，反应完全后，冷却至室温，抽滤，乙醚洗涤3次，收集得到墨绿色固体CMPH 1.18 g，收率为86%。 1 H NMR(400 MHz,CDCl 3 ) δ 10.52(s,1H) 8.41(d, J =15.3 Hz,2H),8.31(d, J =15.0 Hz,2H),7.92(d, J =10.5 Hz,1H),7.81(t, J =7.6 Hz,1H),7.73(t, J =7.4 Hz,1H),7.59(s,1H),7.397.33(m,5H),6.79(s,1H),6.23(s,1H),6.02(s,2H); 13 C NMR(100 MHz,CDCl 3 ) δ 172.4,160.8,154.9,149.5, 143.5,141.0,134.5,133.4,132.4,130.7,129.7,129.0, 128.6,126.6,125.7,122.9,118.2,115.2,113.4,113.2,108.1,58.5。ESI-MS: m/z 412.099 9([M-Br] + ,calcd 412.100 2)。 2.3　光谱测试 2 探针CMPH母液配制:称取一定量的CMPH溶于DMSO，配制成浓度为2 mmol/L的备用储备液，0 ℃保存。移液枪精准移取15 μL探针母液，加入不同pH的PBS缓冲溶液配成浓度为10 μmol/L的测试液，测试探针的紫外和荧光光谱。 2.4　干扰性测试 2 在pH=7.4的条件下，向CMPH(10 μmol/L)中加入各分析物(50 μmol/L)。30 min后，激发波长选择380 nm，测试加入各分析物后溶液荧光强度的变化。 2.5　细胞共聚焦荧光成像 2 在37 ℃、5% CO 2 的湿化气氛下，将人宫颈癌细胞(HeLa)培养于含有10%胎牛血清(FBS)、1%青霉素(100 μg/mL)、1%链霉素(100 μg/mL)的Dulbecco's modified Eagle medium(DMEM)培养基中。将生长状态良好的细胞移至含有10 μmol/L CMPH和0.5 μmol/L商业化染料(Mito-Tracker Green)的DMEM培养基中孵育45 min，用PBS缓冲液洗涤3次。细胞荧光成像在共聚焦显微镜(Leica TCS DIVE/SP8 DIVE)上进行，获得标记细胞的荧光显微镜图像。利用Image J进行图像数据采集和处理。 3　结果与讨论 1 3.1　探针的光学性质 2 分别测试了在不同pH(5.0,6.0,6.5,6.8,7.0,7.5,7.8,8.0,9.0,10.0)的PBS缓冲溶液中CMPH(10 μmol/L)的紫外吸收光谱和荧光发射光谱。如 图2(a) 图2(a) 所示，探针在380 nm处显示出最大吸收峰，当pH值从5.0增加到10.0时，探针CMPH最大吸收峰的峰值也伴随着pH的增大而升高。如 图2(b) 图2(b) 所示，探针CMPH在380 nm激发光的照射下，在460 nm左右显现出最大发射峰，探针的斯托克斯位移为80 nm左右，证明该探针具有背景干扰低的优点。随着pH值的增大，探针的荧光强度呈现逐渐增大的趋势。如 图2(c) 图2(c) 所示，我们对不同pH下探针的荧光强度进行拟合，得出探针的p K a=7.79。如 图2(d) 图2(d) 所示，探针在pH值为6.58.2范围内的响应呈良好的线性关系，线性相关系数为 R 2 =0.99。 3.2　干扰性实验 2 众所周知，细胞内环境是由许多不同的生物分子和离子组成的。为了证实CMPH能够有效地检测细胞内复杂环境中的pH值，在pH=7.4的PBS缓冲液中测试了CMPH在一些阴阳离子和生物信号分子存在下的荧光强度。从 图3 图3 可以看出，探针在分别加入相应的离子和信号分子后，几乎都没有观察到明显的荧光变化，说明CMPH对pH的检测具有较高的选择性。此外，由于羟基的质子化/脱质子化的可逆性，我们分别在pH=5.0和pH=8.0的PBS缓冲液中对探针进行了循环8次的荧光测试。如 图4 图4 所示，CMPH在PBS缓冲溶液中表现出极好的可逆性。 良好的水溶性和稳定性是探针进行生物应用的前提。在pH=7.4的PBS缓冲液中测试了CMPH在不同时间段荧光强度的变化，如 图5 图5 所示，探针在24 h内并无明显的荧光强度变化。同时，在测试过程中并没有发现明显的探针絮状沉淀，表明该探针具有良好的水溶性和稳定性。 3.3　细胞毒性 2 为了评价探针CMPH是否适合细胞成像，我们用MTT法 [ 29 29 ] 测试了CMPH的细胞毒性。即使在使用了20 μmol/L的 CMPH处理HeLa细胞24 h的情况下，仍有88%左右的细胞保持细胞活力，如 图6 图6 所示。结果表明，CMPH具有较低的细胞毒性，对细胞损伤较小，可以进行细胞成像。 3.4　活细胞线粒体共定位荧光成像 2 为了评估探针能否用于活细胞线粒体pH变化的特异性检测，对细胞进行了荧光共聚焦成像。选取HeLa细胞作为实验细胞进行线粒体共定位实验。用含有CMPH(10 μmol/L)和商业染料Mito-Tracker Green(0.5 μmol/L)的培养基共同孵育HeLa细胞30 min。如 图7(a) 图7(a) 所示，在蓝色通道我们观察到强烈的蓝色荧光，并能清楚地看到线粒体的分布，主要分布在细胞质中，且和商业染料标记的线粒体的观察结果一致( 图7(b) 图7(b) )。 图7(c) 图7(c) 为细胞明场。如 图7(d) 图7(d) 所示，我们与商业染料的线粒体定位效果进行对比发现，探针的细胞荧光图像与绿色通道的商业染料染色的细胞荧光图像重叠率很高，重叠系数为 R r =0.92( 图7(e) 图7(e) )。为了进一步说明探针能特异性靶向线粒体，我们截取细胞荧光图像的一段区域进行荧光变化分析，如 图7(f) 图7(f) 所示，在同一位置探针和商业染料的荧光变化几乎同步进行，更直观地揭示了探针CMPH具有良好的线粒体靶向能力。 3.5　细胞pH荧光成像 2 为了验证探针CMPH检测线粒体pH变化的能力，我们用共聚焦显微镜观察了不同pH环境下线粒体的荧光强度。使用不同pH的高K + 缓冲液和尼日利亚菌素(10 μmol/L)培养HeLa细胞30 min。如 图8 图8 所示，我们发现在酸性环境下(pH=5.0)蓝色通道的荧光较弱。随着pH值增大到7.0和9.0，蓝色通道的荧光强度逐渐增强，这与该探针的光学行为表现一致。与其他香豆素类pH荧光探针 [ 30 30 - 34 34 30-34 ] 相比( 表1 表1 )，探针CMPH的p K a=7.79正好对应线粒体弱碱性的pH范围 [ 35 35 ] ，表明CMPH可用于线粒体pH变化的特异性响应。 1 1 Probe 1 1 Structure 1 1 λ ex /nm 1 1 λ em /nm 1 1 Sensitivity(pH) 1 1 Co-localization (mitochondria) 1 1 1 1 1 http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349270&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349279&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349273&type= 1 1 365 1 1 515 1 1 1.593.40 1 1 No 1 1 2 1 1 http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349282&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349290&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349286&type= 1 1 420 1 1 470/583 1 1 2.204.20 1 1 No 1 1 3 1 1 http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349294&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349302&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349298&type= 1 1 385 1 1 455 1 1 7.811.9 1 1 No 1 1 4 1 1 http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349306&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349314&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349310&type= 1 1 400 1 1 515 1 1 3.05.9 1 1 No 1 1 5 1 1 http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349316&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349324&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349319&type= 1 1 322 1 1 405 1 1 2.55.5 1 1 No 1 1 CMPH 1 1 http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349327&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349336&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=21349332&type= 1 1 380 1 1 460 1 1 6.58.2 1 1 Yes 4　结论 1 本文以香豆素为荧光团、3-苄基-2-甲基苯并噻唑溴盐为线粒体定位基团，设计并合成了一种线粒体靶向的pH荧光探针CMPH，该探针对于pH具有较高的特异性和可逆性荧光响应。在探针浓度达20 μmol/L时，24 h后细胞依然具有较高的存活度，表明探针的生物兼容性高。共聚焦荧光成像结果表明，CMPH被成功固定在线粒体中，线粒体的分布清晰可见，并可通过荧光强度的变化监测线粒体pH的波动，揭示了探针CMPH具有监测线粒体pH变化的能力，为线粒体分布及微环境波动的实时监测提供了一种新的性能优良的成像工具。 参考文献: ZHONG W X , RABINOWITZ J D , WHITE E . 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Mater. , 2019 , 2 ( 3 ): 1368 - 1375 . A mitochondria-targeted ratiometric fluorescent pH probe 10.37188/CJL.20200363 O482.31 A 1000-7032(2021)04-0462-08 国家自然科学基金（21778001，21672001）资助项目 Supported by National Natural Science Foundation of China (21778001, 21672001) 2020-11-29 2020-12-31 2021-04-01 2021-10-22 2021 发光学报 04 42