CN114736153A - 一种aie型偶氮酶荧光小分子探针及其制备方法 - Google Patents

一种aie型偶氮酶荧光小分子探针及其制备方法 Download PDF

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CN114736153A
CN114736153A CN202210528981.1A CN202210528981A CN114736153A CN 114736153 A CN114736153 A CN 114736153A CN 202210528981 A CN202210528981 A CN 202210528981A CN 114736153 A CN114736153 A CN 114736153A
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成焕仁
范亚骏
冷静
张培培
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Abstract

本案涉及一种AIE型偶氮酶荧光小分子探针及其制备方法，是以三苯胺衍生物为主体发光单元、偶氮基团为特异性识别基团，通过witting‑horner化反应缩合制得，具有如下结构式：
Figure DDA0003645778880000011
本发明构建了一类结构新颖的AIE型偶氮酶荧光小分子探针，所制备的乏氧酶探针荧光光稳定性好、对乏氧酶选择性识别明显、抗干扰能力强；针对细胞中的偶氮还原酶有显著的吸收信号变化和强烈AIE荧光响应，克服了探针分子较高浓度荧光淬灭的问题，可实现对肿瘤细胞中的乏氧酶实现定量检测，针对细胞中的偶氮还原酶有强烈的吸收和荧光强度及其发射波长变化响应，可实现对肿瘤细胞中的乏氧酶的准确检测。

Description

一种AIE型偶氮酶荧光小分子探针及其制备方法
技术领域
本发明涉及荧光探针技术领域，具体涉及一种AIE型偶氮酶荧光小分子探针及其制备方法。
背景技术
荧光成像技术由于具有可视化、原位、无损等优点，广泛应用于细胞及活体生物成像分析。常用的荧光探针中，小分子荧光探针因其具有容易合成与修饰、光谱易调节、荧光量子产率高、生物相容性良好等优点，备受关注。
有机荧光小分子应用于癌症诊断具有其突出的优点，比如在癌细胞中乏氧是绝大部分实体肿瘤细胞普遍存在的现象，乏氧可以诱导加速生物还原反应和导致细胞内还原酶的表达，如醌还原酶，偶氮还原酶(NZR)以及硝基还原酶。其中，偶氮还原酶是最具代表性的，这种酶催化还原偶氮为氨基反应被证实为一种有效的定位和乏氧成像原理。目前基于NZR机理的识别的荧光探针已被应用到肿瘤诊断上来。然而，相关研究比较单一，大都基于罗丹明和花氰类染料的单分子荧光响应，而基于其它分子的研究相对贫乏---这在一定程度上限制了有机小分子荧光探针在乏氧酶癌症诊断方面的应用。
发明内容
针对现有技术中的不足之处，本发明基于咔唑、噻吩等修饰的三苯胺衍生物与偶氮基团设计并制备了对乏氧酶具有选择性识别的系列荧光小分子探针。
为实现上述目的，本发明提供如下技术方案：
一种AIE型偶氮酶荧光小分子探针，是以三苯胺衍生物为主体发光单元、偶氮基团为特异性识别基团，通过witting-horner化反应缩合制得，具有如下结构式：
Figure BDA0003645778860000021
其中，R1为
Figure BDA0003645778860000022
本发明进一步提供如上所述的AIE型偶氮酶荧光小分子探针的制备方法，包括如下步骤：
S1：制备三苯胺衍生物
S1-1、咔唑修饰三苯胺衍生物：将4,4′-二溴-4″-甲酰基三苯胺，咔唑衍生物加入到反应瓶中，加入碘化亚铜，将1,10-邻菲罗啉和碳酸钾混合物溶解在N,N-二甲基甲酰胺中加入到反应瓶中；氮气保护下加热至160℃反应；反应结束后将反应混合物冷却至室温，倒入冰水中；过滤并干燥，柱层析得固体，即咔唑修饰三苯胺衍生物；
S1-2、联噻吩修饰三苯胺衍生物：将噻吩硼酸酯衍生物和4,4′-二溴-4″-甲酰基三苯胺加入到反应瓶中，加入Pd(PPh3)4、碳酸钾和DMF，将混合物在85℃下加热12h；冷却至室温后，加入水，用1N盐酸调整pH值至弱酸度，有黄色固体沉淀；然后通过柱层析提纯得联噻吩修饰三苯胺衍生物；
S1-3、吡啶乙烯基修饰三苯胺衍生物：氮气保护下，向盛有DMF 100mL的双口瓶中加入4-乙烯基吡啶和4,4′-二溴-4″-甲酰基三苯胺，加入醋酸钯、三(邻甲基苯基)膦和无水磷酸钾，磁力搅拌下，油浴加热至120℃反应24h；反应结束后，将反应液滤入无水甲醇中，抽滤，得到淡黄色固体，即吡啶乙烯基修饰三苯胺衍生物；
S2：氮气保护下，将化合物4,4’-二溴甲基偶氮苯溶于100ml亚磷酸三甲酯中，然后缓慢升温到150℃保温36小时，反应结束后，将反应液倒入石油醚中，大量黄色固体析出，减压抽得，干燥，甲醇重结晶得亚磷酸酯修饰偶氮苯衍生物；
S3：氮气保护下，向含有DMF的反应瓶中加入S1-1或S1-2或S1-3制得的化合物以及S2制得的亚磷酸酯修饰偶氮苯衍生物，加入叔丁醇钾,60-120℃加热回流24小时，反应完成后倒入水中，调节pH至弱酸性，抽滤干燥、纯化得深黄色固体；即得AIE型偶氮酶荧光小分子探针。
本案基于咔唑、噻吩等修饰的三苯胺衍生物与偶氮基团设计并制备了对乏氧酶具有选择性识别的系列荧光探针分子。探针分子与乏氧酶作用在乏氧酶的还原作用下，分子共轭基团被破坏并生成具有AIE效应的小分子，通过这种选择性识别后产生的发光波长和发光强度的变化实现对生物体内乏氧状态的鉴定，进而可以根据浓度曲线计算乏氧程度并以此推断细胞或组织的癌变情况。本案制备的材料光稳定性好、发光效率高、探针分子与乏氧酶作用后生成的新的分子具有AIE效应，克服了较高浓度下探针荧光淬灭的问题，同时，识别前后明显的发光强度和发光蓝移现象可以满足复杂环境下检测的需要。
与现有技术相比，本发明的有益效果是：本发明构建了一类结构新颖的AIE型偶氮酶荧光小分子探针，所制备的乏氧酶探针荧光光稳定性好、对乏氧酶选择性识别明显、抗干扰能力强；针对细胞中的偶氮还原酶有显著的吸收信号变化和强烈AIE荧光响应，克服了探针分子较高浓度荧光淬灭的问题，可实现对肿瘤细胞中的乏氧酶实现定量检测，针对细胞中的偶氮还原酶有强烈的吸收和荧光强度及其发射波长变化响应，可实现对肿瘤细胞中的乏氧酶的准确检测。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为探针分子SFX-ON的紫外吸收谱图。
图2为探针分子SFX-ON对乏氧酶(AZR)的识别增强荧光光谱图。
图3为探针分子SFX-ON对乏氧酶的选择性识别柱状图(1-Na+、2-K+、3-Ca2+、4-Mg2+、5-H202、6-GSH、7-DTT、8-Arg、9-Try、10-Cys、11-His、12-Leu、13-Glu、14-SOD、15-HSA、16-BSA、17-GOx、18-AZR、探针浓度：2μM，激发波长为400nm)。
图4为探针分子SFX-ON在THF-H2O中比率荧光AIE效果图。
图5为探针分子SFX-ON对乏氧酶的荧光识别机理图。
图6为探针分子SFX-ON与癌细胞的荧光成像图(a、乏氧状态的细胞；b、乏氧状态的细胞+探针分子SFX-ON)。
具体实施方式
下面将结合附图对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
此外，下面所描述的本发明不同实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互结合。
实施例1：
步骤1
Figure BDA0003645778860000051
氮气保护下，将化合物4,4’-二溴甲基偶氮苯4g(11mmol)、溶于100ml亚磷酸三甲酯中，然后缓慢升温到150℃保温36小时，色谱检测跟踪反应进程，反应结束后，将反应液倒入50ml石油醚中，大量黄色固体析出，减压抽得，干燥，甲醇重结晶得一淡黄色固体ON-1，4.2g，产率：90％。1H NMR(300MHz,CDCl3):δ8.96-8.93(d,J＝9.0Hz,4H),7.36-7.33(d,J＝9.0Hz,4H),3.64(s,12H),3.03(d,4H)。
步骤2：
Figure BDA0003645778860000052
氮气保护下，将4,4′-二溴-4″-甲酰基三苯胺(SX-1，5.0g,9.5mmol)，3,6-二叔丁基-9-氢咔唑(KZ-1，5.8g,20.9mmol)，碘化亚铜(0.72g,3.78mmol)，1,10-邻菲罗啉(1.5g，8.3mmol)和碳酸钾(3.5g,20.9mmol)混合物溶解在N,N-二甲基甲酰胺(50mL)中。氮气保护下加热至160℃反应24h。反应结束后将反应混合物冷却至室温，倒入冰水中(100mL)。过滤并干燥，柱层析得黄色固体SKD-1，产率：85％。1H NMR(300MHz,CDCl3):9.93(s,1H),8.18(s,4H),7.87(d,J＝8.52Hz,2H),7.63(d,J＝8.49Hz,4H),7.54–7.45(m,12H),7.31(d,J＝8.19Hz,2H),1.50(s,36H)。
步骤3：
Figure BDA0003645778860000061
氮气保护下，向含有DMF 100ml的250mL两口瓶中，加入化合物ON-11.8g(4.2mmol)，化合物SKD-1 6.8g(8.4mmol)，叔丁醇钾1.0g(10mmol)，60-120℃油浴锅中反应48小时。反应结束后冷却至室温，将反应液倒入100ml冰水中，用稀盐酸条件pH至弱酸性，有大量黄色固体析出，减压抽滤，柱层析(二氯甲烷/石油醚)得化合物SDK-ON 6.6g，产率：86％。1H NMR(300MHz,CDCl3):δ8.95-8.92(d,J＝9.0Hz,4H),8.18(s,8H),7.88(d,J＝8.50Hz,4H),7.64(d,J＝8.48Hz,8H),7.56–7.45(m,24H),7.38-7.34(d,J＝9.0Hz,4H),7.32(d,J＝8.20Hz,4H),6.92-6.86(d,J＝9.0Hz,4H),1.50(s,72H)。
实施例2：
步骤1
Figure BDA0003645778860000062
氮气保护下，将化合物SX-1(2.5g，5.8mmol)、Pd(PPh3)4(0.022g，0.18mmol)、碳酸钾(0.85g，8mmol)、SF-1(3.75g，13mmol)溶于DMF中(80ml)，85℃下加热12h；冷却至室温后，加入水(100mL)，用1N盐酸调整pH值至弱酸度，黄色固体沉淀量，在50℃的真空干燥箱中干燥24小时。然后通过柱层析(用二氯甲烷/甲醇＝9/1洗脱)进行更严格的纯化，得到黄色固体SFX-1，2.0g，产率：90％。1H NMR(CDCl3,ppm):δ9.83(s,1H),7.81-7.78(d,2H,J＝9.0Hz),7.72-7.70(d,4H,J＝6.0Hz),7.54-7.53(d,2H,J＝3Hz),7.49-7.48(d,2H,J＝3Hz),7.36-7.33(q,4H),7.21-7.13(d,4H),7.12-7.08(q,4H)。
步骤2
Figure BDA0003645778860000071
参考例1步骤3得一黄色固体，产率：88％。1H NMR(300MHz,CDCl3):8.96-8.93(d,J＝9.0Hz,4H),δ7.81-7.78(d,4H,J＝9.0Hz),7.72-7.70(d,8H,J＝6.0Hz),7.54-7.53(d,4H,J＝3Hz),7.49-7.48(d,4H,J＝3Hz),7.36-7.33(m,12H),7.21-7.13(d,8H),7.12-7.08(q,8H)。
实施例2：
步骤1：
Figure BDA0003645778860000072
氮气保护下，向盛有DMF 100mL的双口瓶中加入4-乙烯基吡啶(1.0mL，8mmol)，化合物SX-1(1.5g，3.5mmol)，醋酸钯(0.0225g，0.10mmol)，三(邻甲基苯基)膦(0.06g，0.20mmol)和无水磷酸钾(0.7g，3mmol)，磁力搅拌下，油浴加热至120℃反应24h。反应结束后，将反应液滤入无水甲醇中，抽滤，得到橙红色固体，真空干燥，粗产物用柱层析提纯，梯度洗脱，得到橙红色固体BEX-1，2.25g，产率75％。1H NMR(CDCl3,300MHz):δ9.87(s,1H)，8.57(d,J＝6.00Hz,4H),7.74(d，J＝6.00Hz，2H)，7.50(d,J＝9.00Hz,4H)，7.37(d,J＝9.00Hz,4H),7.32(s,2H),7.14-7.19(m,4H),6.95(d,J＝18.00Hz,4H)。
步骤2：
Figure BDA0003645778860000081
参考例1步骤3得黄色固体BEX-ON，产率：90％。1H NMR(300MHz,CDCl3):δ8.96-8.93(d,J＝9.0Hz,4H),8.57(d,J＝6.00Hz,8H),7.74(d，J＝6.00Hz，4H)，7.50(d,J＝9.0Hz,8H),7.42(d,J＝9.0Hz,8H),7.36-7.32(d,J＝9.0Hz,8H),7.14-7.19(m,8H),6.98(d,J＝18.00Hz,8H),6.92-6.86(d,J＝9.0Hz,4H)。
以实施例2制得的材料SFX-ON为例，进行荧光和紫外光谱测试：将材料配置成10-5～10-6mol/L浓度的二氯甲烷溶液，采用紫外吸收光谱仪和荧光光谱仪分别测定材料是吸收光谱和发射光谱(图1-2)。
本发明设计的反应条件温和、反应高效、操作简便、适用性广，为丰富和开发红外乏氧酶检测提供了新的思路。
实施例4：选择性荧光识别实验：
2μg/mL的荧光探针SFX-ON在10mM PBS(pH＝7.4)溶液中分别与金属离子(Na+、K+、Ca2+、Mg2+，2mM)、氧化还原分子(AA、H2O2、GSH、DTT，2mM)、氨基酸(Arg、Try、Cys、His、Leu、Glu，2mM)和蛋白质(BSA、HSA，2mg/mL；SOD、GOx，2μg/mL)以及2μM的AZR在37℃反应半小时，然后检测其荧光变化情况。从图3可以发现，除AZR外，其它溶液的荧光信号都没有发生明显变化。结果表明，探针SFX-ON对乏氧相关偶氮还原酶具有特异响应能力。图4是荧光探针SFX-ON在THF-H2O中比率荧光AIE效果图，可以看到由左至右绿色荧光越明显。图5是荧光探针SFX-ON对乏氧酶的荧光识别机理图，探针分子与乏氧酶作用在乏氧酶的还原作用下，分子共轭基团被破坏并生成具有AIE效应的小分子。
实施例5：荧光细胞成像实验：
本发明进一步考察了乏氧探针SFX-ON用于细胞荧光成像的研究。35mm的培养皿接种癌细胞后，分别在1％氧气和常氧环境中孵育24小时，然后与2μg/mL探针SFX-ON反应一段时间并用AZR进行细胞核染色，最后用共聚焦荧光显微镜进行成像研究，以未用探针处理的细胞作为对照，激发波长为405nm。如图6所示，乏氧状态的细胞当没有与探针SFX-ON结合时，只有很暗的黄光(图6a)；而当乏氧状态下的细胞与乏氧探针反应后再用激光进行照射时，则能观察到很明显的绿色荧光(图6b)。
尽管本发明的实施方案已公开如上，但其并不仅仅限于说明书和实施方式中所列运用，它完全可以被适用于各种适合本发明的领域，对于熟悉本领域的人员而言，可容易地实现另外的修改，因此在不背离权利要求及等同范围所限定的一般概念下，本发明并不限于特定的细节和这里示出与描述的图例。

Claims (2)

1.一种AIE型偶氮酶荧光小分子探针，是以三苯胺衍生物为主体发光单元、偶氮基团为特异性识别基团，通过witting-horner化反应缩合制得，具有如下结构式：
Figure FDA0003645778850000011
其中，R1为
Figure FDA0003645778850000012
2.如权利要求1所述的AIE型偶氮酶荧光小分子探针的制备方法，其特征在于，包括如下步骤：
S1：制备三苯胺衍生物
S1-1、咔唑修饰三苯胺衍生物：将4,4′-二溴-4″-甲酰基三苯胺，咔唑衍生物加入到反应瓶中，加入碘化亚铜，将1,10-邻菲罗啉和碳酸钾混合溶解在N,N-二甲基甲酰胺中加入到反应瓶中；氮气保护下加热至160℃反应；反应结束后将反应混合物冷却至室温，倒入冰水中；过滤并干燥，柱层析得固体，即咔唑修饰三苯胺衍生物；
S1-2、联噻吩修饰三苯胺衍生物：将噻吩硼酸酯衍生物和4,4′-二溴-4″-甲酰基三苯胺加入到反应瓶中，加入Pd(PPh3)4、碳酸钾和DMF，将混合物在85℃下加热12h；冷却至室温后，加入水，用1N盐酸调整pH值至弱酸度，有黄色固体沉淀；然后通过柱层析提纯得联噻吩修饰三苯胺衍生物；
S1-3、吡啶乙烯基修饰三苯胺衍生物：氮气保护下，向盛有DMF 100mL的双口瓶中加入4-乙烯基吡啶和4,4′-二溴-4″-甲酰基三苯胺，加入醋酸钯、三(邻甲基苯基)膦和无水磷酸钾，磁力搅拌下，油浴加热至120℃反应24h；反应结束后，将反应液滤入无水甲醇中，抽滤，得到淡黄色固体，即吡啶乙烯基修饰三苯胺衍生物；
S2：氮气保护下，将化合物4,4’-二溴甲基偶氮苯溶于100ml亚磷酸三甲酯中，然后缓慢升温到150℃保温36小时，反应结束后，将反应液倒入石油醚中，大量黄色固体析出，减压抽得，干燥，甲醇重结晶得亚磷酸酯修饰偶氮苯衍生物；
S3：氮气保护下，向含有DMF的反应瓶中加入S1-1或S1-2或S1-3制得的化合物以及S2制得的亚磷酸酯修饰偶氮苯衍生物，加入叔丁醇钾,60-120℃加热回流24小时，反应完成后倒入水中，调节pH至弱酸性，抽滤干燥、纯化得深黄色固体；即得AIE型偶氮酶荧光小分子探针。
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