CN107056827A - 一种基于bodipy类单线态氧荧光探针 - Google Patents
一种基于bodipy类单线态氧荧光探针 Download PDFInfo
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Abstract
本发明公开了一种基于BODIPY类单线态氧荧光探针，本发明对单线态氧的检测具有高灵敏、高选择性、在中性环境中响应快的特点。该探针本身没有荧光，与单线态氧响应后，荧光显著增强，荧光强度与单线态氧的浓度呈良好的线性关系，可用于定量检测，且其它活性氧物质不干扰其检测。制备的荧光探针具有经典的BODIPY光谱特性，可基于荧光增强或色度增强检测化学样品、生物样品或医学样品中的汞离子，并适合荧光成像。
Description
技术领域
本发明属于荧光探针领域，具体设计一种基于BODIPY荧光染料的增强型的单线态氧荧光探针的制备方法和应用。
背景技术
在生物体系中，单线态氧（1O2）是重要的活性氧物种。1O2具有高的化学活性和较大的扩散半径，它能穿透线粒体膜或核膜，造成DNA的损伤；可与组蛋白作用，组氨酸残基经1O2氧化作用，产生一种新的光产物。并且，它的多少与细胞损伤、死亡成正比关系；可氧化半胱氨酸、组氨酸、色氨酸、蛋氨酸和酪氨酸残基，使蛋白质丧失功能；可使酶蛋白分子发生分子内或分子间交联，导致构型改变而失活或暴露出某些隐蔽的肽键而易遭蛋白酶的水解，从而造成人的衰老。上述这些作用将损坏有机体，包括对细胞膜的损伤致变作用、再生的改变等，在生物体系中可能引起许多蛋白质光氧化疾病。因此，1O2一方面具有较强的毒性，造成机体的损伤，另一方面又是生物体免疫系统的主要杀菌剂，还可用于癌症的光动力疗法。
由于1O2反应活性高，寿命很短，在水溶液中，寿命约为10-6-10-5 s，故其分析测定仍然是一项难题。1O2采用的分析方法有分光光度法、电子顺磁共振法、磷光光度法、色谱法、化学发光法等。这些方法或有的容易受到共存离子、溶剂等因素的影响，并且无法满足生理条件下检测的需要；或有的所用仪器相对昂贵，操作程序也相对复杂等缺点。这些对了解体内1O2的功能机制显著地受到影响。虽然用磷光法能够直接监测1O2，但缺乏实用性，因为其发射具有非常低的光致发光量子产率（PLQYs≈10-6）。因此，设计具有高的灵敏度和宽的动力学范围来检测生物体中1O2的探针已成为分析研究的热点之一。
在各种各样的探针中，荧光探针由于具有高的灵敏度和低的检出限，并且在生物成像应用方面有很大优势，是目前公认的检测1O2的最佳方法。通过连接荧光团与1O2捕获基团而设计的荧光探针已经有一些报道，但其灵敏度不高，有些是需在碱性环境中，检测范围受到限制。这些报道的荧光团有荧光素、聚芴、菁等，捕获基团有蒽及衍生物、组氨酸等。在1O2和捕获基团之间发生[2＋4]环加成反应后，荧光发生变化。但是这些探针中，以BODIPY类染料作为设计平台的单线态氧的荧光探针还鲜有报道。而BODIPY类荧光染料具有良好的光化学物理性能，如摩尔消光系数高，荧光量子产率高，荧光光谱峰宽窄、光热化学稳定性高、染料光谱性质稳定、染料分子结构中不含离子电荷、较小的分子质量与较低的细胞毒性等优良的性质。基于此，我们设计了一种基于BODIPY类的荧光增强型的单线态氧的荧光探针，以实现在生理条件下对1O2的高选择性、高灵敏度的识别及检测。
发明内容
本发明的目的在于提供一种单线态氧荧光探针的制备方法及应用，从而快速准确、灵敏地检测光动力疗法中光敏剂产生的1O2的浓度。
为了实现上述目的本发明采用如下技术方案：
一种针对单线态氧检测的荧光探针C1，其结构式为：
所述的C1荧光探针，其设计特征在于将分子中蒽与BODIPY染料巧妙的合成连起来，利用蒽把BODIPY的荧光淬灭，整个分子几乎没有荧光；当与单线态氧作用后，发生[2＋4]环加成反应后，BODIPY的荧光恢复。荧光从无到有，从而可以实现可视化检测单线态氧。
所述的单线态氧荧光探针的制备方法，其特征在于包括以下两个步骤：
第一步：准确称取9，10-2甲基-2醛基蒽（1.17 g，5.0 mmol）溶于干燥的 CH2Cl2（500mL），在氮气的气氛下，慢慢滴入新蒸 2,4-二甲基吡咯（1.1 m L，12.5 mmol），再加入0.1mL三氟乙酸作为催化剂，反应液由澄清逐渐变成血红色，室温搅拌下反应10小时。
第二步：用针筒注入溶于干燥四氢呋喃的二氯二氰基苯醌（DDQ）（1.13 g，5.0mmol），继续室温搅拌下反应 0.5 h。再在冰水浴条件下，慢慢加入三乙胺（10.0 m L，71.9 mmol），加完之后继续搅拌15min，再慢慢滴加三氟化硼乙醚（10.0 m L，79.6 mmol），撤掉冰水浴，室温下反应2.5 h。反应结束后，加入250 m L水，用二氯甲烷（3×120 m L）萃取，合并二氯甲烷相，用无水硫酸钠干燥，过滤，旋干溶剂。粗产物用（二氯甲烷：石油醚=1：5）洗脱纯化，得到产物C1为紫红色固体(23 %)。
本发明提供的探针C1是基于BODIPY染料荧光增强型的荧光探针，具有经典BODIPY的光谱特性，即光稳定性好，较高的摩尔消光系数、较高的荧光量子产率和较低的细胞毒性等特性，并且吸收和发射波长均在可见光区，适合荧光显微镜分析检测生物样品中单线态氧的定量和定性分析与荧光成像。
本发明提供的荧光探针可以用于化学样品、生物样品或医学样品中单线态氧的定量分析与荧光成像。
本发明提供的探针，突出的优点是：
本发明提供的荧光探针只有两步合成，并为一锅反应，合成简单，合成条件较温和；
本探针的设计是基于单线态氧的[2＋4]环加成反应，所以选择性高，其中羟基自由基（HO·）、超氧阴离子自由基（O2 -·）、双氧水（H2O2）、次氯酸根（ClO-）、过氧亚硝基（ONOO-）、一氧化氮（NO）对探针响应很小，选择性好；
本探针是荧光增强型的，与未加入单线态氧的探针溶液，增强倍数达到27倍，灵敏度高；
本探针C1-H2O2-ClO-系统在中性环境中荧光强度与单线态氧的浓度呈现良好的线性关系；
本探针C1对单线态氧可以快速响应，当反应时间在15分钟内时，荧光强度迅速增强，之后荧光强度几乎不变。如此快的响应时间（表明此反应的活性大），能应用于实时检测。
本探针结构中由于蒽基团的光致电子转移（PET）导致探针本身的荧光被淬灭，当与单线态氧反应后，发生[2＋4]环加成，蒽的共轭结构被破坏，PET终止，BODIPY的荧光迅速恢复，荧光迅速增强，在365nm的手持荧光灯下肉眼可见，可用于可视化检测单线态氧。
附图说明
图1 探针C1的合成路线
图2 探针C1 (5 µM)对各种活性氧物种的响应。1.blank, 2.HO·, 3. O2 -·, 4.H2O2, 5. ClO-, 6. ONOO-, 7. NO, 8. 1O2. 1O2.: 100 μM NaOCl + 0.5 mM H2O2; NO: 100mM 1-hydroxy-2-oxo-3-(3-amino-propyl)-3-methyl-1-triazene (NOC-13, NO供体);H2O2 : 100 mM H2O2 ; O2 -·: 100 mM KO2；ONOO-: 100 mM 3-morpholinosydnonimine(SIN-1, ONOO-供体); ClO-: 100 mM NaOCl; HO·: 100 mM H2O2 + 100 mM (NH4)2Fe(SO4)2
图3 探针C1与50 mM H2O2 在不同浓度的NaOCl缓冲溶液（Tris-HCl, pH=7.4）中的荧光光谱图。
图4 在525nm处荧光强度与单线态氧浓度呈线性关系。
图5 荧光探针C1(5 µM)与1O2（100 µM）的荧光强度随时间变化曲线。
具体实施方式
下面的实施例只是用于详细说明本发明，并不以任何方式限制发明的范围。
实施例探针的合成：
准确称取9，10-2甲基-2醛基蒽（1.17 g，5.0 mmol）溶于干燥的 CH2Cl2（500 mL），在氮气的气氛下，慢慢滴入新蒸 2,4-二甲基吡咯（1.1 m L，12.5 mmol），再加入0.1 mL三氟乙酸作为催化剂，反应液由澄清逐渐变成血红色，室温搅拌下反应10小时。然后用针筒注入溶于干燥四氢呋喃的二氯二氰基苯醌（DDQ）（1.13 g，5.0mmol），继续室温搅拌下反应 0.5 h。再在冰水浴条件下，慢慢加入三乙胺（10.0 m L，71.9 mmol），加完之后继续搅拌15min，再慢慢滴加三氟化硼乙醚（10.0 m L，79.6 mmol），撤掉冰水浴，室温下反应2.5 h。反应结束后，加入250 m L水，用二氯甲烷（3×120 m L）萃取，合并二氯甲烷相，用无水硫酸钠干燥，过滤，旋干溶剂。粗产物用（二氯甲烷：石油醚=1：5）洗脱纯化，得到产物C1为紫红色固体(23%)。
光谱测试：
在试管中加入 0.03 mL探针C1母液（5×10-4mol/L），0.27 mL乙醇，和 2.7 mL pH值为7.4的Tris-HCl缓冲溶液，混合均匀，然后加入0.5 mM H2O2 在不同浓度的NaOCl缓冲溶液，反应15 min后进行光谱测定。激发光波长为450 nm, 激发光狭缝宽度为5.0 nm, 发射峰狭缝宽度为5.0 nm。图3可知，探针C1对单线态氧具有较高的灵敏度。并且在2×10−6 mol/L到1×10−4 mol/L浓度范围内，探针在525 nm处的荧光强度与1O2的浓度呈现良好的线性关系（图4），线性关系为F=89.52973+4.25406 c，相关系数为R = 0.997，这表明在较大的浓度范围内可以作定量检测。
当在荧光探针C1(5 µM)中分别加入100 mM 羟基自由基（HO·）、超氧阴离子自由基（O2 -·）、双氧水（H2O2）、次氯酸根（ClO-）、过氧亚硝基（ONOO-）、一氧化氮（NO），没有引起明显的荧光增强，只有当加入单线态氧（1O2）时荧光显著增强（图2）。
考察了荧光探针C1(5 µM)与1O2（100 µM）的反应时间，探针C1与1O2反应大约15分钟后（图5），其荧光强度达到饱和，说明探针C1在生物样品实时检测及荧光成像应用方面将会有较大优势。
Claims (7)
1.一种基于BODIPY类单线态氧荧光探针，其结构如下：
权利要求1所述荧光探针C1的制备方法，其特征步骤包含以下步骤：
准确称取9，10-二甲基-2-醛基蒽（1.17 g，5.0 mmol）溶于干燥的 CH2Cl2（500 mL），在氮气的气氛下，慢慢滴入新蒸的 2,4-二甲基吡咯（1.1 m L，12.5 mmol），再加入0.1 mL三氟乙酸作为催化剂，反应液由澄清逐渐变成血红色，室温搅拌下反应10小时。
2.然后用针筒注入溶于干燥四氢呋喃的二氯二氰基苯醌（DDQ）（1.13 g，5.0mmol），继续室温搅拌下反应 0.5 h。
3.再在冰水浴条件下，慢慢加入三乙胺（10.0 m L，71.9 mmol），加完之后继续搅拌15min，再慢慢滴加三氟化硼乙醚（10.0 m L，79.6 mmol），撤掉冰水浴，室温下反应2.5 h。
4.反应结束后，加入250 m L水，用二氯甲烷（3×120 m L）萃取，合并二氯甲烷相，用无水硫酸钠干燥，过滤，旋干溶剂。
5.粗产物用（二氯甲烷：石油醚=1：5）洗脱纯化，得到产物C1为紫红色固体(23%)。
6.权利要求1所述荧光探针在中性溶液中能够检测单线态氧，灵敏度高、选择性好，并且检测的线性范围宽。
7.权利要求1所述荧光探针用于化学样品、生物样品或医学样品中单线态氧的定量分析与荧光成像。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108997404A (zh) * | 2018-10-11 | 2018-12-14 | 广东工业大学 | 一种光敏剂、制备方法及其应用 |
| CN111233905A (zh) * | 2018-11-29 | 2020-06-05 | 中国科学院大连化学物理研究所 | 一种选择性检测溶液相中单线态氧的荧光探针及应用 |
| CN111362977A (zh) * | 2018-11-29 | 2020-07-03 | 中国科学院大连化学物理研究所 | 一种双分子极性化合物及合成与应用 |
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| HISATO SUNAHARA ET AL.,: ""Design and Synthesis of a Library of BODIPY-Based Environmental Polarity Sensors Utilizing Photoinduced Electron-Transfer-Controlled Fluorescence ON/OFF Switching"", 《J. AM. CHEM. SOC.》 * |
| STEFANO BANFI ET AL.,: ""Synthesis and photo-physical properties of a series of BODIPY dyes"", 《TETRAHEDRON》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108997404A (zh) * | 2018-10-11 | 2018-12-14 | 广东工业大学 | 一种光敏剂、制备方法及其应用 |
| CN111233905A (zh) * | 2018-11-29 | 2020-06-05 | 中国科学院大连化学物理研究所 | 一种选择性检测溶液相中单线态氧的荧光探针及应用 |
| CN111362977A (zh) * | 2018-11-29 | 2020-07-03 | 中国科学院大连化学物理研究所 | 一种双分子极性化合物及合成与应用 |
| CN111362977B (zh) * | 2018-11-29 | 2021-08-17 | 中国科学院大连化学物理研究所 | 一种双分子极性化合物及合成与应用 |
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