CN111057085A - 一种靶向线粒体的过氧亚硝酸根阴离子荧光探针的制备及应用 - Google Patents
一种靶向线粒体的过氧亚硝酸根阴离子荧光探针的制备及应用 Download PDFInfo
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Abstract
本发明公开了一种靶向线粒体的过氧亚硝酸根阴离子(OONO‑)荧光探针的制备及应用。三苯基膦阳离子的引入使该探针可以精确定位至线粒体，被过氧亚硝酸根阴离子激活后在活细胞中具有优异的成像效果。本发明还提供了一种可功能化激活式荧光探针的通用制备策略，可以通过click反应引入不同的功能基团，使目标探针具有各种可预期的优良性能；在酚羟基上醚化不同的生物响应性基团，使其可以特异性识别不同的生物信号分子，释放荧光并成像，这种激活式的策略可以显著降低背景干扰，从而提高检测灵敏度。
Description
技术领域
本发明涉及一种靶向线粒体的过氧亚硝酸根阴离子荧光探针的制备及应用，属于荧光探针技术领域。
背景技术
ONOO-是一氧化氮与超氧阴离子自由基快速反应的产物，主要在线粒体内产生。由于OONO-具有很强的氧化性和亲核性，可以与蛋白质、脂质体和核酸等生物分子发生反应，心血管疾病、神经退行性疾病、代谢性疾病等疾病的发病机理都与其密切相关。因此，检测细胞内的OONO-对这些疾病的早期诊断具有重要意义。荧光探针以其无创监测、操作方便、灵敏度高、时空分辨率高等优点，被认为是荧光成像技术的有力工具。吡喃腈(DCM)类荧光探针具有高荧光量子产率、长波长荧光发射以及斯托克斯位移大等优点，但传统的DCM类荧光探针不具有靶向特性，并且受到其低水溶性的限制，导致该类探针在实际生物应用中存在一定的困难，高灵敏度的成像仍然是一个挑战。因此，基于DCM荧光团设计一个靶向线粒体、在水环境中分散性好且可以灵敏检测OONO-的荧光探针具有重大意义。
发明内容
本发明的目的就是为了解决现有技术中存在的上述问题，提供了一种靶向线粒体的过氧亚硝酸根阴离子荧光探针的制备及应用，合成路线见附图1。
本发明提供的一种靶向线粒体的过氧亚硝酸根阴离子荧光探针，具有如mito- PNFlu-6.2所示结构。
作为优选，化合物1和Flu-6.2所示结构的化合物的摩尔比为1：1.2，四氢呋喃和水的体积比为4：1，反应温度为室温，反应时间为12h。
作为优选，化合物mitoFlu-6.2和PN-trigger所示结构的化合物的摩尔比为1：3，反应溶剂为丙酮，反应温度为65°C。
本发明提供了一种样品配制方法，即先将含有Flu-6.2所示结构的化合物的二甲基亚砜(DMSO)溶液和含有F127的DMSO溶液混合均匀，再快速稀释到缓冲液中。
mito-PNFlu-6.2所示结构的化合物荧光关闭，和线粒体内过表达的OONO-作用后，生成具有强烈荧光的mitoFlu-6.2所示结构的化合物，实现线粒体靶向荧光探针的可激活性。
在PBS/ DMF体积比为7：3的混合溶液中，mito-PNFlu-6.2所示结构的化合物可迅速对4eq的OONO-完全响应，溶液的pH为7.4，含有F127 (0.3%)。
本发明还提供了一种可功能化特异性激活式荧光探针的通用制备策略，可以通过click反应将不同的功能基团引入Flu-6.2所示结构的化合物，使其具有各种可预期的优良性能；在酚羟基上醚化不同的生物响应性基团，使其可以特异性识别不同的生物信号分子，实现荧光从“关闭”到“开启”的转变，显著提高检测灵敏度三苯基膦阳离子的引入使该探针可以精确定位至线粒体。
附图说明
图1靶向线粒体的过氧亚硝酸根阴离子荧光探针的合成路线。
图2以罗丹明B为标准品，Flu-6.2荧光量子产率的测定。
图3靶向线粒体的过氧亚硝酸根阴离子荧光探针在OONO-刺激下的转化情况。
图4靶向线粒体的过氧亚硝酸根阴离子荧光探针在刺激响应前后的荧光发射。
图5靶向线粒体的过氧亚硝酸根阴离子荧光探针在不同刺激物存在下的荧光发射。
图6靶向线粒体的过氧亚硝酸根阴离子荧光探针在细胞中的定位情况。
图7靶向线粒体的过氧亚硝酸根阴离子荧光探针分子结构式。
具体实施方式
以下结合附图和实施例对本发明做出进一步详细说明，但所举实施例不作为本发明的限定，凡在本发明的主体设计思想上经过无创造性的替代或变换所获得的其他实施例，都在本发明的保护范围之内。
实施例一：合成mito-PNFlu-6.2所示结构的化合物，合成路线见附图1。
将化合物Flu-6.2 (0.131g, 0.25mmol)和化合物1 (0.184g, 0.2mmol)溶于四氢呋喃/水(4mL/1mL)的混合溶液中。加入抗坏血酸钠(4.0mg, 0.02mmol)和无水硫酸铜(3.2mg, 0.02mmol)。反应混合物在室温下搅拌12h。反应结束后，用二氯甲烷萃取，无水硫酸钠干燥，旋蒸除去溶剂。以二氯甲烷和甲醇为洗脱剂进行柱层析，得到棕黄色固体化合物mitoFlu-6.2 (0.213g，73.8%)。1H NMR (400 MHz, CDCl3) δ (ppm) 8.93 (s, 1H), 8.59(s, 1H), 8.21 (s, 1H), 7.81–7.56 (m, 15H), 7.42–7.21 (m, 5H), 6.94 (s, 1H),6.63–6.47 (m, 2H), 4.90 (s, 2H), 4.81–4.52 (m, 4H), 4.29–4.07 (m, 6H), 3.92–3.48 (m, 28H), 3.40–3.22 (m, 12H), 1.25 (s, 9H). 13C NMR spectrum (100 MHz,CDCl3) δ (ppm) 166.80, 162.17, 156.60, 152.62, 152.54, 152.01,151.18, 146.04,140.51, 137.98, 135.64, 134.85, 134.82, 133.88, 133.78, 130.21, 130.09,129.20, 127.58, 127.31, 124.13, 123.70, 123.07, 119.13, 118.27, 117.74,117.44, 110.69, 107.01, 106.46, 100.62, 77.37, 72.40, 71.86, 70.87, 70.55,70.50, 70.40, 70.25, 70.20, 69.68, 68.73, 63.89, 63.82, 59.50, 59.00, 49.18,35.86, 35.49, 29.72, 29.60, 25.35, 24.82. HRMS (MALDI): m/z [M] calcd forC76H88Cl2N6O16P+ 1441.5366; found 1443.5374。
将mitoFlu-6.2 (0.2g, 0.139mmol)、K2CO3 (0.058g, 0.417mmol)加入丙酮(20mL)中，搅拌升温至65°C。氮气保护下逐滴滴加含有化合物trigger-PN (0.083g, 0.28mmol)的丙酮溶液。滴加完毕后继续反应4h。旋蒸除去溶剂后，以二氯甲烷和甲醇为洗脱剂进行柱层析得到黄色固体化合物mito-PNFlu-6.2 (0.146g, 63.3%)。1H NMR (400 MHz, CDCl3,) δ(ppm) 8.72 (s, 1H), 8.39 (t, J = 4.0 Hz, 1H), 8.22 (s, 1H), 7.85 (d, J = 7.6Hz, 2H), 7.80–7.59 (m, 15H), 7.59–7.46 (m, 5H), 7.29 (s, 2H), 7.00 (s, 1H),6.81–6.69 (m, 2H), 5.10 (s, 2H), 4.90 (t, J = 5.6 Hz, 2H), 4.74 (d, J = 5.6Hz, 2H), 4.65 (t, J = 5.6 Hz, 2H), 4.19 (m, 6H), 3.94–3.72 (m, 10H), 3.71–3.55 (m, 14H), 3.53–3.48 (m, 4H), 3.42–3.20 (m, 12H), 1.36 (s, 12H), 1.28 (s,9H). 13C NMR (100 MHz, CDCl3) δ (ppm) 166.61, 162.18, 156.05, 152.72, 152.67,152.15, 152.05, 145.93, 140.63, 138.96, 138.20, 134.96, 134.84, 134.81,133.91, 133.80, 132.51, 130.41, 130.20, 130.08, 129.29, 128.07, 127.56,124.15, 123.90, 120.42, 119.12, 118.26, 117.26, 116.17, 110.88, 107.45,107.03, 100.78, 84.24, 83.90, 77.37, 75.12, 72.42, 71.87, 70.89, 70.56,70.51, 70.43, 70.40, 70.27, 70.19, 69.68, 68.86, 67.20, 63.93, 63.86, 60.69,59.00, 49.09, 35.90, 35.46, 29.57, 25.55, 25.03, 24.91. HRMS (MALDI): m/z [M]calcd for C89H105BCl2N6O18P+ 1657.6688; found 1659.6707。
以罗丹明B（乙醇中荧光量子产率为89%）为标准品，研究Flu-6.2所示结构的化合物的荧光量子产率。溶液的pH为7.4，含有DMSO (5%), F127 (0.3%)。结果如附图2所示，计算得Flu-6.2荧光量子产率为56.57%。
探究mito-PNFlu-6.2所示结构的化合物在OONO-刺激下的转化情况。结果如附图3所示，经OONO-刺激响应后，mito-PNFlu-6.2转变为mitoFlu-6.2。
探究mito-PNFlu-6.2所示结构的化合物在刺激响应前后的荧光发射。我们在PBS/DMF (7/3)的混合溶液中对化合物mito-PNFlu-6.2 (5μM)进行测试。溶液的pH为7.4，含有F127 (0.3%)。结果如附图4所示，刺激响应前荧光完全被抑制，刺激响应后荧光恢复。
探究mito-PNFlu-6.2所示结构的化合物对不同刺激物响应的选择性。我们在PBS/DMF (7/3)的混合溶液中对化合物mito-PNFlu-6.2 (5μM)进行测试。溶液的pH为7.4，含有F127 (0.3%)。结果如附图5所示，mito-PNFlu-6.2可以对OONO-特异性响应，表现为刺激响应后，荧光恢复，而对其余的刺激物没有响应，荧光仍处于关闭状态。
探究mito-PNFlu-6.2所示结构的化合物靶向细胞线粒体的能力。结果如附图6所示，刺激响应后，化合物mito-PNFlu-6.2和商业的线粒体探针荧光高度重合，皮尔森系数高达95%，说明化合物mito-PNFlu-6.2可以很好地定位到线粒体。
以上所述仅为本发明的优选实施例，不应当用于限制本发明的保护范围，凡在本发明的主体设计思想上做出的无实质意义的替代或变换(例如，以化合物mitoFlu-6.2为底物，分别醚化不同的刺激响应性基团)，均在本发明的保护范围之内。
Claims (7)
1.一种靶向线粒体的过氧亚硝酸根阴离子荧光探针的制备方法见附图1。
2.根据权利要求1所述的制备方法，化合物1和Flu-6.2所示结构的化合物的摩尔比为1：1.2，四氢呋喃和水的体积比为4：1，反应温度为室温，反应时间为12h。
3.根据权利要求1所述的制备方法，mitoFlu-6.2所示结构的化合物和PN-trigger所示结构的化合物的摩尔比为1：2，反应溶剂为丙酮，反应温度为65°C。
4.本发明提供了一种样品配制方法，即先将含有Flu-6.2所示结构的化合物的二甲基亚砜(DMSO)溶液和含有F127的DMSO溶液混合均匀，再快速稀释到缓冲液中。
5.mito-PNFlu-6.2所示结构的化合物荧光关闭，和线粒体内过表达的OONO-作用后，生成具有强烈荧光的mitoFlu-6.2所示结构的化合物，实现线粒体靶向荧光探针的可激活性。
6.在PBS/N,N-二甲基甲酰胺(DMF)体积比为7：3的混合溶液中，mito-PNFlu-6.2所示结构的化合物可迅速对4eq的OONO-完全响应，溶液的pH为7.4，含有F127 (0.3%)。
7.本发明提供了一种可功能化特异性激活式荧光探针的通用制备策略，可以通过click反应将不同的功能基团引入Flu-6.2所示结构的化合物，使其具有各种可预期的优良性能；在酚羟基上醚化不同的生物响应性基团，使其可以特异性识别不同的生物信号分子，实现荧光从“关闭”到“开启”的转变，显著提高检测灵敏度。
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