CN112645918A - 一种aie型香豆素衍生物荧光探针及其在氰离子检测中的应用 - Google Patents
一种aie型香豆素衍生物荧光探针及其在氰离子检测中的应用 Download PDFInfo
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Abstract
本发明公开了具体涉及一种AIE型香豆素衍生物荧光探针及其在氰离子检测中的应用，属于有机小分子生物荧光探针技术领域，所述荧光探针的化学名称为（E）‑8‑（（（二苯亚甲基）肼基）甲基）‑7‑羟基‑4‑甲基‑2H‑苯并吡喃‑2‑酮(C‑BH)。该荧光探针表现出极大的斯托克斯位移（248 nm），能有效解决因荧光自吸导致在生物应用方面缺陷，荧光分析研究表明该化合物可用于氰离子的高灵敏检测，其检测限为0.05µM。
Description
技术领域
本发明属于有机小分子生物荧光探针技术领域，具体涉及一种AIE型香豆素衍生物荧光探针及其在氰离子检测中的应用。
背景技术
氰化物在地表水中的存在不仅是由工业废物造成的，也是由生物过程造成的。由于氰化物在生理系统中的极端毒性及其在环境中的广泛存在，人们对发展检测氰化物的方法给予了相当大的关注。一方面，氰化物在人的血液中可与细胞色素C氧化酶形成稳定的复合物，从而抑制该酶的功能，导致细胞毒性缺氧和细胞窒息。另一方面，氰化物引起的厌氧代谢导致乳酸在血液中积累。缺氧和乳酸酸中毒的联合作用扰乱了中枢神经系统的稳定，导致呼吸停止和甚至死亡。据报道，氰化物的LD50(估计对50％的暴露人口是致命的剂量)为2500-5000mg/L，根据世界卫生组织，饮用水中氰化物的最高可接受浓度为1.9μM。由于氰化物的广泛存在和剧毒性，其精准、高灵敏及快速检测主要还是依靠荧光分析法。设计结构新颖的荧光探针分子，丰富其荧光成像方式并提高成像性能，已逐渐成为荧光成像技术发展过程中的热点与难点。
与传统的荧光探针相比，AIE分子生物探针的简单设计和荧光开启特性提供了水介质中特异性分析物和生物过程的直接可视化，具有更高的灵敏度和更好的准确性。不同剂型和表面功能的AIE点生物探针在量子点和小分子染料上表现出吸收率大、发光度高、生物相容性好、无随机闪烁、耐光漂白等先进特性，这些特性使肿瘤细胞检测、长期细胞示踪和肿瘤成像成为可能。最近的研究极大地扩展了AIE荧光剂的生物应用范围，并为荧光生物探针的设计提供了新的策略。预计，AIE生物探针的未来发展将将单光子或多光子荧光与其他形式(如磁共振成像)或功能(如治疗)相结合，以充分展示其作为新一代治疗诊断试剂的潜力。与此同时，分子生物学的发展将提供更特异性的生物受体，这将有助于开发具有高选择性和高灵敏度的下一代AIE生物探针，用于分子传感和成像。近几年，对AIE分子的研究取得了很大的进展，如硅杂环戊二烯及其衍生物、四苯基乙烯(TPE)衍生物以及其它具有AIE现象的分子，在生物检测领域得到了广泛的应用。尽管如此，用于检测氰化物的AIE型荧光探针仍很缺乏，这类探针亟待开发。
发明内容
针对目前可用于氰化物检测的AIE型荧光探针缺乏的现状，本发明提供了一种AIE型香豆素衍生物荧光探针及其制备方法和应用。
为了实现上述发明目的，本发明采用以下技术方案：
一种AIE型香豆素衍生物荧光探针，其结构式如Ⅰ所示：
该荧光探针的化学名称为(E)-8-(((二苯亚甲基)肼基)甲基)-7-羟基-4-甲基-2H-苯并吡喃-2-酮，分子式为C24H18N2O3。
上述荧光探针的制备方法是：在酸性条件下，7-羟基-4-甲基-8-乙烯基-2H-苯并吡喃-2-酮和(二苯基亚甲基)肼反应得到目标探针。
具体地：将中间体7-羟基-4-甲基-8-乙烯基-2H-苯并吡喃-2-酮溶于有机溶剂中，然后加入(二苯基亚甲基)肼，再加入适量醋酸，搅拌数小时，薄层色谱法点板跟踪至反应结束后，抽滤后得到橙黄色固体(E)-8-(((二苯亚甲基)肼基)甲基)-7-羟基-4-甲基-2H-苯并吡喃-2-酮。
其中，中间体7-羟基-4-甲基-8-乙烯基-2H-苯并吡喃-2-酮由以下方法制备得到：
将乌洛托品先溶解在醋酸中，搅拌反应，再加入7-羟基-4-甲基-2H-苯并吡喃-2-酮，加热回流，用薄层色谱法监测进程，之后，加入盐酸溶液，再加热，待反应完成后冷却至室温，用乙醚萃取，有机层减压蒸干，将残渣倒入冰水中，得到淡黄色固体(纯)产品7-羟基-4-甲基-8-乙烯基-2H-苯并吡喃-2-酮。
进一步地，所述有机溶剂选自甲苯、乙腈、二氯乙烷、二氯甲烷、三氯甲烷、四氯化碳、正己烷、四氢呋喃、甲醇或乙醇。优选为乙醇。
上述制备方法中，反应结束后的后处理方式并无特别的限定，本领域技术人员可依据物料的理化性质，结合公知常识的分离手段，采用常规的物理分离手段来实现目标产物的分离，优选的技术方案为过滤法，更进一步优选，选用减压抽滤。
上述制备方法中，通过薄层色谱法监测反应终点，反应时间无特别的限定。
本发明还提供了上述荧光探针在氰离子检测中的应用。
一种氰离子检测试剂，包括上述荧光探针。
进一步地，所述氰离子检测试剂为体内氰离子检测试剂。
本发明通过7-羟基-4-甲基-8-乙烯基-2H-苯并吡喃-2-酮中的醛基与(二苯基亚甲基)肼中的氨基通过希夫碱反应，形成C＝N双键，从而提供可以用于氰离子检测的作用位点。
有益效果:
1、本发明首次提供了一种具有AIE性能的香豆素类化合物荧光探针，丰富了氰化物类荧光分子探针的种类，为有机分析和光化学提供了新型的探针分子，可广泛应用于荧光分析或检测领域。
2、该新型荧光分子探针实现了对氰离子的高灵敏检测，其检测限为0.05μM。
3、该荧光探针分子可以实现在HeLa细胞中对氰离子含量检测和成像研究。
附图说明
图1为探针分子C-BH在不同含水量的DMSO-H2O混合液中的荧光图。
图2为探针分子C-BH在DMSO溶液中紫外及荧光图及斯托克斯位移。
图3为探针分子C-BH的DMSO溶液对其他不同类型干扰物的荧光图。
图4为探针分子C-BH的DMSO溶液对不同浓度氰离子的荧光图。
图5为探针分子C-BH的DMSO溶液对氰离子检测动力学图。
图6为探针分子C-BH在HeLa细胞中对氰离子的成像图。图中的A1、B1、C1和D1是细胞的绿色通道和蓝色通道的叠加图，随着CN-的增加，叠加图由绿色的光变成蓝色的光，A2、B2、C2和D2是细胞的明场图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步详细说明，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。实施例中未注明具体条件的实验方法及未说明配方的试剂均为按照本领域常规条件。
实施例1
AIE型香豆素衍生物荧光探针的制备：
第1步、化合物7-羟基-4-甲基-8-乙烯基-2H-苯并吡喃-2-酮的制备
将乌洛托品(1,9627g,14mmol)先溶解在15mL HAc中，在75℃下搅拌反应约30min后，再加入7-羟基-4-甲基-2H-苯并吡喃-2-酮(1.0568g,6mmol)加热至75℃回流，用薄层色谱法监测进程，7h后，加入15mL的20％HCl，再加热约45min，反应完成后冷却至室温，用乙醚萃取，有机层减压蒸干，将残渣倒入冰水(30mL)中，得到淡黄色固体(纯)产品。(注：7-羟基-4-甲基-2H-苯并吡喃-2-酮购买于南京晴文生物科技有限公司。)
1H NMR(400MHz,Chloroform-d)δ12.25(s,1H),10.65(d,J＝0.6Hz,1H),7.76(d,J＝9.0Hz,1H),6.93(dd,J＝9.0,0.6Hz,1H),6.23(t,J＝1.2Hz,1H),2.45(d,J＝1.3Hz,3H).
第2步、化合物(E)-8-(((二苯亚甲基)肼基)甲基)-7-羟基-4-甲基-2H-苯并吡喃-2-酮的制备
称取中间体7-羟基-4-甲基-8-乙烯基-2H-苯并吡喃-2-酮(0.8mmol,0.1632g)和(二苯基亚甲基)肼(1mmol,0.1963g)溶于乙醇中，再加入一滴醋酸提供酸性环境，90℃搅拌反应，约6h后，反应液中逐渐析出橙黄色固体，冷却，过滤，用冷得乙醇洗涤得到探针(E)-8-(((二苯亚甲基)肼基)甲基)-7-羟基-4-甲基-2H-苯并吡喃-2-酮(C-BH)。
1H NMR(400MHz,DMSO-d6)δ12.05(s,1H),9.20(s,1H),7.70(t,J＝8.1Hz,3H),7.62–7.44(m,6H),7.32(dd,J＝6.7,3.0Hz,2H),6.78(d,J＝8.9Hz,1H),6.24(s,1H),2.36(s,3H).13C NMR(101MHz,DMSO-d6)δ169.45,162.63,159.49,158.18,154.32,153.84,136.61,135.54,132.02,130.53,129.77,129.26,129.21,129.17,128.51,113.72,112.22,111.31,105.72,18.80.ESI-MS m/z:[probe]-calcd for C24H20N2O3 381.13；Found 381.2.
下面对探针分子C-BH的荧光性质及其在氰离子检测中的应用进行进一步研究。
1、探针分子C-BH在不同含水量的DMSO-H2O混合液中的荧光性能
测试仪器：日立F7100型分子荧光光谱仪。
实验方法为：将实施例1制得的探针分子C-BH溶解于DMSO中得到1mM的探针母液，常温保存，用探针母液分别配制成0％、10％、30％、50％、70％、75％、80％、85％、90％含水量的DMSO-H2O混合液，浓度均为0.1mM。
测量时分别移取3mL不同含水量的DMSO-H2O混合液到1cm比色皿中先后进行荧光光谱测试，如图1所示。当含水量由0％增加到85％时，由于水的诱导聚集，C-BH的发射信号持续增强，表现出典型的AIE特征；当含水量为90％时，C-BH容易沉淀，因此观察到相对较差的发射性能。故选用85％含水量的探针溶液用于C-BH的后续测试。
2、探针分子C-BH的紫外吸收光谱及荧光光谱性质测试
测试仪器：PE 950s型紫外光谱仪，日立F7100型分子荧光光谱仪。
实验方法为：将实施例1制得的探针分子C-BH溶解于DMSO溶液中得到1mM的探针母液，常温保存。实验测定中用DMSO和H2O将溶液稀释成0.01mM的标准液进行测试。
测量时移取3mL探针的DMSO溶液到1cm比色皿中先后进行紫外吸收光谱及荧光光谱测试，如图2所示。结果表明：探针C-BH最强紫外吸收峰出现在320nm左右，荧光发射峰出现在568nm左右，斯托克位移达到248nm。这么大的斯托克位移，可以有效的克服因荧光自吸收而在生命体中难以应用的缺陷，实现探针在生命体中的应用。
3、探针分子C-BH对其他干扰离子的荧光图
测试仪器：日立F100型分子荧光光谱仪。
实验方法为：将实施例1制得的探针分子C-BH溶解于DMSO中得到1mM的探针母液，常温保存。取四丁基氰化铵、ZnSO4、AgNO3、Co(NO3)2、CuSO4、Fe2(SO4)3、PbNO3、NaNO2、NaNO3、KH2PO4、NaHSO4、Na2S、KCl、CaCl2用二次水配置成0.01M母液。实验测定中将探针溶液稀释成1μM的标准溶液进行测试。
测量时移取3mL探针的DMSO溶液到1cm比色皿中分别滴加35μM的半胱氨酸、ZnSO4、AgNO3、Co(NO3)2、Fe2(SO4)3、Pb(NO3)2、NaNO2、NaNO3、KH2PO4、KCl、CH3COONa、半胱氨酸进行荧光测试。结果如图3所示。结果表明：探针C-BH对氰离子表现出明显的荧光增强现象，而对于一些生物体内常见的金属阳离子和酸根阴离子几乎没有什么影响，进一步说明探针C-BH具有优异的选择性，能够应用于生物体内。
4、C-BH的DMSO溶液对四丁基氰化铵的定量分析
测试仪器：日立F7100型分子荧光光谱仪。
实验方法为：将实施例1制得的探针分子C-BH溶解于DMSO中得到1mM的探针母液，常温保存。四丁基氰化铵用二次水配置成0.01M母液，实验测定中将探针溶液稀释成1μM的标准溶液进行测试。
采用标准加入法测试探针分子对氰离子的荧光响应，移取3mL的探针母液(1μM)至比色皿中，每次加入0.5μL的氰离子检测荧光强度变化，氰离子含量加到35μM不再继续加入，如图4所示，随着氰离子含量的增加，在438nm处的荧光峰强度不断增强，因此，该探针对氰离子有较高的灵敏度，可用于生物体内微量氰离子的检测。
5、探针分子C-BH在氰离子存在下的动力学实验
测试仪器：日立F7100型分子荧光光谱仪。
实验方法为：将实施例1制得的探针分子C-BH溶解于DMSO中得到1mM的探针母液，常温保存。四丁基氰化铵用二次水配置成0.01M母液。实验测定中将溶液稀释成1μM的标准溶液进行测试。
移取3mL的探针母液(1μM)至比色皿中，设置荧光激发波长为320nm，分别测试探针，探针+氰离子溶液在不同的时间(0分钟、1分钟、2分钟、3分钟、5分钟、10分钟、15分钟、20分钟、25分钟)荧光强度的变化，如图5所示。实验结果表明，开始探针溶液的荧光强度随时间的增加而增强，探针+氰离子在10min内荧光强度达到最高值，后荧光强度趋于稳定，说明该探针响应迅速且稳定性较好。
6、探针分子C-BH在HeLa细胞中对氰离子的成像研究。
实验方法为：将实施例1制得的探针分子C-BH溶解于DMSO中得到1mM的探针母液，常温保存。实验测定中用DMSO和H2O将溶液稀释成0.01mM的标准液进行测试。
为了证明探针在生物系统的实际应用，在共聚焦荧光显微镜下对细胞进行了在不同pH值下的生物荧光成像实验。将HeLa细胞接到培养皿中在37℃条件下培养24h，然后将C-BH标准液(10μM)加入到培养皿中，加入不同浓度的氰离子(5、10、15μM)继续孵育1小时后进行荧光成像，如图6所示。实验结果表明探针分子C-BH在随着氰离子浓度增加，荧光在不断增强。这些结果表明，探针C-BH可以作为检测细胞内氰离子的荧光标签进入细胞，因而具有在生物体内检测氰离子的潜力。
Claims (6)
1.一种AIE型香豆素衍生物荧光探针，其特征在于：其结构式如Ⅰ所示：
2.权利要求1所述AIE型香豆素衍生物荧光探针的制备方法，其特征在于：该荧光探针的制备方法如下式所示：
7-羟基-4-甲基-8-乙烯基-2H-苯并吡喃-2-酮和(二苯基亚甲基)肼在酸性环境下反应，制得所述荧光探针。
3.根据权利要求2所述的制备方法，其特征在于：
所述7-羟基-4-甲基-8-乙烯基-2H-苯并吡喃-2-酮通过下式所示方法制备得到：
7-羟基-4-甲基-2H-苯并吡喃-2-酮和乌洛托品在酸性条件下反应，制得7-羟基-4-甲基-8-乙烯基-2H-苯并吡喃-2-酮。
4.权利要求1所述的AIE型香豆素衍生物荧光探针在氰离子检测中的应用。
5.一种氰离子检测试剂，其特征在于：包括权利要求1所述的AIE型香豆素衍生物上述荧光探针。
6.根据权利要求5所述的氰离子检测试剂，其特征在于：所述氰离子检测试剂为体内氰离子检测试剂。
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| CN107235946A (zh) * | 2017-06-20 | 2017-10-10 | 济南大学 | 一种谷胱甘肽荧光探针及其制备方法和应用 |
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