CN115124555B - 基于苝酰亚胺衍生物的乏氧酶荧光材料及其制备方法 - Google Patents
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Abstract
本案涉及一种基于苝酰亚胺衍生物的乏氧酶荧光材料及其制备方法，该材料以苝酰亚胺为主体发光单元、偶氮衍生物作为修饰基团，通过酰胺化反应/偶联化反应制得，其具有如下结构式：其中，R1为长链烷基。本发明选择苝酰亚胺衍生物作为发光主体，通过化学修饰在苝的弯位引入偶氮基团以调整分子的发光性能，在苝的酰胺位引入长链烷烃以调节分子的溶解性能和对细胞等组织的穿透能力；探针分子与乏氧酶作用，偶氮基团被还原从而影响分子主体发光强度发生强烈的变化，发光波长发生蓝移，通过这种选择性识别的变化进而表达细胞或者组织内乏氧酶的存在，借助荧光浓度滴定可以定量测定乏氧酶浓度，从而实现对生物体内的癌变情况的诊断。
Description
技术领域
本发明涉及荧光探针技术领域，具体涉及一种基于苝酰亚胺衍生物的乏氧酶荧光材料及其制备方法。
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，可通过各种不同的原理(比如PET机理、ICT机理和FRET机理)构建灵敏度高、选择性好、响应快的荧光探针，从而实现对特定目标物质的响应性识别。
乏氧是绝大部分实体瘤中的普遍现象，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。目前基于这NZR机理的识别研究有所报道，然而，相关研究还不是很丰富，大都基于罗丹明和花氰类染料。
发明内容
针对现有技术中的不足之处，本发明基于偶氮衍生物并结合苝酰亚胺发光单元，制备一种乏氧酶荧光材料。
为实现上述目的，本发明提供如下技术方案：
一种基于苝酰亚胺衍生物的乏氧酶荧光材料，该材料以苝酰亚胺为主体发光单元、偶氮衍生物作为修饰基团，通过酰胺化反应/偶联化反应制得，其具有如下结构式：
其中，R1为长链烷基。
本发明提供如上所述的基于苝酰亚胺衍生物的乏氧酶荧光材料的制备方法，包括如下步骤：
S1：向反应瓶中加入1-溴苝四甲二酸酐、氨基化合物，乙醇做溶剂升温到85℃油浴锅中反应48小时，反应结束后，分离提纯得中间体1；
S2：氮气保护下，向反应瓶中加入中间体1、4-醛基苯硼酸/氯化二苯基膦钯、碳酸钾、邻甲基三苯基膦以及四丁基氯化铵，加入溶剂DMSO，于100～120℃反应48h，分离提纯得中间体2；
S3：将4,4,’-二溴甲基偶氮苯溶解在一定量的亚磷酸甲酯中，升温到150℃油浴锅中反应24小时，反应结束后，自然冷却，加入适量的石油醚分离提纯得中间体3；
S4：氮气保护下，向反应瓶中加入中间体2和中间体3，叔丁醇钾作碱，DMF作溶剂，60-120℃加热24h，反应结束后，用二氯甲烷/甲醇分离提纯得最终产物，即得。
进一步地，所述氨基化合物为C4～C20的长链烷基胺。
进一步地，所述S1中1-溴苝四甲二酸酐、氨基化合物的摩尔比为1:2.2～2.8。
与现有技术相比，本发明的有益效果是：本发明选择苝酰亚胺衍生物作为发光主体，通过化学修饰在苝的弯位引入偶氮基团以调整分子的发光性能，在苝的酰胺位引入长链烷烃以调节分子的溶解性能和对细胞等组织的穿透能力。探针分子与乏氧酶作用，偶氮基团被还原从而影响分子主体发光强度发生强烈的变化，发光波长发生蓝移，通过这种选择性识别的变化进而表达细胞或者组织内乏氧酶的存在，借助荧光浓度滴定可以定量测定乏氧酶浓度，从而实现对生物体内的癌变情况的诊断。本案制备的材料光稳定性好、发光效率高、为其在复杂生物体环境的诊疗效果提供了保证。
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为本案制得的探针分子CHR-C对乏氧酶的(AZR)的紫外吸收谱图。
图2为本案制得的探针分子CHR-C对乏氧酶的(AZR)的识别增强荧光光谱图。
图3为本案制得的探针分子CHR-C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。)。
图4为为本案制得的探针分子CHR-C用于肝细胞的荧光成像图(a非乏氧条件下探针分子与肝细胞的成像图；b乏氧条件下探针分子与乏氧细胞成像图)。
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：
向250mL两口瓶中，加入化合物1-溴代苝二酸酐8g(17mmol)，正丁胺3g(41mmol)溶于150ml无水乙醇中，85℃油浴锅中反应48小时。通过薄层层析板检测反应进程，反应结束后，减压除去溶剂，乙醇重结晶得红色固体CHR-1，8.4g，产率：85％。δ8.95(s,1H),8.35-8.29(d,J＝9.0Hz,3H),8.20-8.16(d,J＝9.0Hz,3H),3.24(m,12H),1.54-1.30(m,8H),0.92(m,6H)。
步骤2
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CHR-A4.2g,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3：
氮气保护下，向250mL两口瓶中，加入化合物溴代萘酐CHR-1 6.5g(12mmol)，4-醛基苯硼酸81.g(13mmol)，氯化二苯基膦钯(25mg)，碳酸钾1.8g(12mmol)，邻甲基三苯基膦0.8g，四丁基氯化铵(1g)溶于120ml DMSO中，100-125℃油浴锅中反应48小时。通过薄层层析板检测反应进程，反应结束后，冷却至室温，将反应液倒入150ml冰水中，减压抽滤，乙醇重结晶，得化合物NDK-CH，5.6g，产率：79％。1H NMR(300MHz,CDCl3):δ8.95(s,1H),8.35-8.26(m,5H),8.20-8.16(d,J＝9.0Hz,3H),7.83-7.80(d,J＝9.0Hz,2H),3.24(m,12H),1.54-1.30(m,8H),0.93(m,6H)。
步骤4：
氮气保护下，向含有DMF 90ml的250mL两口瓶中，加入化合物CHR-A1.6g(4mmol)，化合物CHR-2 5g(8.2mmol)，叔丁醇钾1.0g(10mmol)，60-120℃油浴锅中反应48小时。通过薄层层析板检测反应进程，反应结束后冷却至室温，将反应液倒入150ml冰水中，用稀盐酸条件pH至弱酸性，有大量绿色固体析出，减压抽滤，柱层析(二氯甲烷/甲醇)得化合物CHR-C4.2g，产率：76％。1H NMR(300MHz,CDCl3):δ8.95(s,2H),8.35-8.26(m,6H),8.20-8.16(d,J＝9.0Hz,6H),8.0-7.91(d,J＝9.0Hz,8H),7.65-7.62(d,J＝9.0Hz,8H),6.95-6.92(d,J＝9.0Hz,4H),2.84(m,8H),1.54-1.30(m,16H),1.02(m,12H)。
对实施例1制得的材料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。
实施例2：荧光识别实验：
2μg/mL的荧光探针CHR-C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CHR-C对乏氧相关偶氮还原酶具有特异响应能力。
实施例3：荧光细胞成像实验：
本发明进一步考察了乏氧探针CHR-C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CHR-C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CHR-C处理的细胞作为对照，激发波长为500nm。如图4所示，不管常氧还是乏氧状态的细胞，当没有与探针CHR-C反应时，500nm激光照射后不能观测到红光。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红色荧光(图4)。结果表明本案构建的探针确实能够用于乏氧成像。
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)
1.一种基于苝酰亚胺衍生物的乏氧酶荧光材料，其特征在于，该材料以苝酰亚胺为主体发光单元、偶氮衍生物作为修饰基团，通过酰胺化反应/偶联化反应制得，其具有如下结构式：。
2.如权利要求1所述的基于苝酰亚胺衍生物的乏氧酶荧光材料的制备方法，其特征在于，包括如下步骤：
步骤1：
；
步骤2：
；
步骤3：
；
步骤4：
。
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