CN115557878A - 一种吲哚菁类衍生物及其制备和应用 - Google Patents
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Abstract
本发明公开了一种吲哚菁类衍生物及其制备和应用,所述吲哚菁类衍生物具有如下结构如下式:
Description
技术领域
本发明涉及生物分析技术领域,具体的涉及一种吲哚菁类衍生物及其制备和应用。
背景技术
近年来,随着荧光探针技术的不断发展,吲哚菁类化合物在荧光探针中的应用也越来越多。吲哚菁作为一种常见的荧光探针,具有良好的生物相容性和较大的斯托克斯位移等特点。吲哚菁类荧光探针由于具有供体-π-受体结构常表现出分子内电荷转移效应,并产生较大的斯托克斯位移。另外,由于吲哚菁中含有一个易被质子化的氮原子,因此基于吲哚菁类的荧光探针往往具备对pH检测的能力。
吲哚菁类荧光探针具有广泛的生物活性,包括抗癌、抗病毒、抗菌、抗炎、抗艾滋病和抗糖尿病等,常用于治疗窗口的荧光探针进行精确、准确和选择性检测。由于吲哚菁类化合物具有低毒性、极佳的组织穿透水平等优点,可在临床实践中作为医学成像和诊断试剂,因此在制药领域中得到广泛研究。基于吲哚菁设计的荧光探针往往具有近红外的光谱性质,这类荧光探针具有生物组织穿透力强、生物背景荧光干扰小、发光范围广、荧光性质好、分子标记结构稳定、膜通透性高等特点,因此常用于器官染色和细胞间物质变化的监测。
发明内容
本发明目的是为荧光分析法提供一种吲哚菁类衍生物及其制备和应用,该探针易于合成并且敏感性、选择性好。
为实现上述目的,本发明采用了以下的技术方案:
本发明所述的一种吲哚菁类衍生物,具有以下结构式:
其中R是以下之一:
所述的吲哚菁类衍生物的制备方法,包括如下步骤:依次向60mL乙醇中2,3,3-三甲基-4,5-苯并吲哚、甲醛类化合物和哌啶进行反应,然后将反应溶液加热并在氮气气氛下回流,冷却至室温后减压去除溶剂,并通过快速柱色谱法获得产物。
所述的2,3,3-三甲基-4,5-苯并吲哚、甲醛类化合物和哌啶的添加量分别为10mmol,12mmol及0.1mmol。
所述的加热是将反应溶液升温到120℃。
所述回流的时间为18h。
所述的甲醛类化合物是3-氰基苯甲醛、3-羧基苯甲醛及香豆素-6-甲醛之一。
所述的吲哚菁类衍生物在pH荧光探针上的应用。
所述pH的检测范围为1.0-7.0。
本发明的有益效果是:
本发明所述的一种吲哚菁类pH荧光探针,所述荧光探针以吲哚菁为pH敏感基团,通过吲哚菁在酸性条件下质子化使分子在不用的pH中荧光发射强度不同。
所述探针对pH响应敏感,并且有着10倍以上的荧光强度变化。
所述探针有着较大的斯托克斯位移,可以使探针在荧光分析时具有背景干扰低、检测灵敏度高等优点。
所述探针有着良好的水溶性,以及优秀的抗干扰性和荧光可逆性。
所述探针通过结合不同的基团,pH检测范围可以由1.0至6.0,满足酸性条件下细胞成像的需要,可作为细胞成像荧光探针使用。
附图说明
图1是实施例3制备的吲哚菁类pH荧光探针A在不同pH下荧光发射光谱。
图2是实施例3制备的吲哚菁类pH荧光探针A不同pH下荧光发射光谱的S型拟合。
图3是实施例3制备的吲哚菁类pH荧光探针B在不同pH下荧光发射光谱。
图4是实施例3制备的吲哚菁类pH荧光探针B不同pH下荧光发射光谱的S型拟合。
图5是实施例3制备的吲哚菁类pH荧光探针C在不同pH下荧光发射光谱。
图6是实施例3制备的吲哚菁类pH荧光探针C不同pH下荧光发射光谱的S型拟合。
图7是实施例3制备的吲哚菁类pH荧光探针C在pH 3.0和5.0时干扰下的荧光发射光谱。
图8是实施例3制备的吲哚菁类pH荧光探针C在pH 3.0和5.0之间的荧光重复性。
图9是实施例3制备的吲哚菁类pH荧光探针C在不同pH培养下HeLa细胞共聚焦成像。
具体实施方式
下面结合具体附图对本发明做进一步的说明。
检测所用仪器为:BrukerAVANCE III HD500型核磁共振仪(TMS为内标,氘代DMSO为溶剂),岛津RF-5301PC型荧光分光光度计,雷磁PHSJ-4A型精密pH计。
所述荧光探针均由2,3,3-三甲基-4,5-苯并吲哚与相应的吸电子或给电子基团通过Knoevenagel缩合反应制备,所述荧光探针的反应的方程式如下:
实施例1
本实施例是制备所述的吲哚菁类衍生物中R为A的一个应用实施例。
所述的吲哚菁类衍生物中R为A的合成:向60mL乙醇中加入2.09g(10mmol)2,3,3-三甲基-4,5-苯并吲哚和1.57g(12mmol)3-氰基苯甲醛和0.01mL(0.1mmol)哌啶。然后将反应溶液120℃加热并在氮气气氛下回流18小时。冷却至室温后,减压去除溶剂,并通过快速柱色谱法(二氯甲烷:甲醇=20:1)获得化合物A,黄色固体1.54g(产率:48%)。1H NMR(500MHz,DMSO-d6)δ8.44(d,J=1.7Hz,1H),8.22–8.13(m,2H),8.04(d,J=8.2Hz,1H),7.97(d,J=8.5Hz,1H),7.90(d,J=16.2Hz,1H),7.86–7.79(m,2H),7.69–7.58(m,3H),7.52(ddd,J=8.1,6.7,1.1Hz,1H),1.64(s,6H).13C NMR(126MHz,DMSO)δ185.09,151.36,140.08,137.77,135.19,133.01,132.72,132.57,131.48,130.51,129.95,129.47,128.60,127.21,125.26,123.53,121.96,120.60,119.15,112.55,54.69,22.27.HRMS calculatefor C23H18N2[M+H]+:323.41,found:323.15。
实施例2
本实施例是制备所述的吲哚菁类衍生物中R为B的一个应用实施例。
所述的吲哚菁类衍生物中R为B的合成:向60mL乙醇中加入2.09g(10mmol)2,3,3-三甲基-4,5-苯并吲哚和1.80g(12mmol)3-羧基苯甲醛和0.01mL(0.1mmol)哌啶。然后将反应溶液120℃加热并在氮气气氛下回流18小时。冷却至室温后,减压去除溶剂,并通过快速柱色谱法(二氯甲烷:甲醇=20:1)获得化合物B,黄色固体2.97g(产率:87%)。1H NMR(500MHz,DMSO-d6)δ8.36(s,1H),8.19(d,J=8.4Hz,1H),8.11(d,J=7.8Hz,1H),8.04(d,J=8.1Hz,1H),7.99–7.90(m,3H),7.81(d,J=8.5Hz,1H),7.66–7.55(m,2H),7.55–7.47(m,2H),1.66(s,4H).13C NMR(126MHz,DMSO)δ185.29,167.62,151.38,140.12,136.86,136.64,132.51,132.25,132.08,130.34,129.95,129.65,129.43,128.98,128.57,127.18,125.18,123.46,120.87,120.58,54.63,22.50.HRMS calculate for C23H19NO2[M+H]+:341.41,found:314.15。
实施例3
本实施例是制备所述的吲哚菁类衍生物中R为C的一个应用实施例。
所述的吲哚菁类衍生物中R为C的合成:向60mL乙醇中加入2.09g(10mmol)2,3,3-三甲基-4,5-苯并吲哚和2.09g(12mmol)香豆素-6-甲醛和0.01mL(0.1mmol)哌啶。然后将反应溶液120℃加热并在氮气气氛下回流18小时。冷却至室温后,减压去除溶剂,并通过快速柱色谱法(二氯甲烷:甲醇=20:1)获得化合物C,黄色固体1.53g(产率:42%)。1H NMR(500MHz,DMSO-d6)δ8.31(dd,J=7.7,1.6Hz,1H),8.28(d,J=16.3Hz,1H),8.20(dd,J=8.3,1.1Hz,1H),8.14(d,J=9.5Hz,1H),8.05(dd,J=8.3,1.3Hz,1H),7.98(d,J=8.5Hz,1H),7.87(d,J=8.4Hz,1H),7.77(dd,J=7.6,1.5Hz,1H),7.66–7.59(m,2H),7.52(ddd,J=8.1,6.8,1.2Hz,1H),7.46(t,J=7.7Hz,1H),6.60(d,J=9.6Hz,1H),1.65(s,6H).13C NMR(126MHz,DMSO)δ185.18,159.95,151.59,151.42,145.02,139.93,132.57,129.95,129.48,128.71,128.62,127.20,125.24,124.95,124.08,123.52,121.98,120.72,119.77,116.84,54.59,22.32.HRMS calculate for C25H19NO2[M+H]+:366.53,found:366.14。
实施例4
选取实施例1制备的化合物A进行性能测试。取28.7mg的A用5mL甲醇溶解后加入100mL棕色容量瓶后用水定容,探针母液浓度为1mM,使用0.1mol/L柠檬酸-柠檬酸钠缓冲液稀释母液至相应的pH后,获得浓度为10μM的光谱测定溶液。光谱测量时将3.0mL溶液倒入1cm光程的石英比色皿中。荧光发射光谱使用390nm波长,激发狭缝宽度为5nm,发射狭缝宽度为10nm。如图1所示,化合物A的最大发射波长为500nm,荧光强度随着pH值降低而下降并呈现红移趋势。通过比较pH 1.0和pH 4.0条件下500nm处的荧光发射强度,我们发现化合物A的荧光发射强度增加了10倍以上。
实施例5
选取实施例1制备的化合物A进行对pH与荧光强度关系图中点的S型拟合。如图2所示,截取pH 1.6至pH 3.4范围内pH与荧光强度关系图进行线性拟合,化合物A的荧光与pH变化呈良好的线性关系,R2达到0.981,pKa为2.50。
实施例6
选取实施例2制备的化合物B按照实施例4的方法进行性能测试。如图3所示,化合物B的最大发射波长为540nm,荧光强度随着pH值降低而下降并呈现红移趋势。通过比较pH1.0和pH 4.0条件下540nm处的荧光发射强度,我们发现化合物B的荧光发射强度增加了10倍以上。
实施例7
选取实施例2制备的化合物B进行对pH与荧光强度关系图中点的S型拟合。如图4所示,截取pH 1.4至pH 3.2范围内pH与荧光强度关系图进行线性拟合,化合物B的荧光与pH变化呈良好的线性关系,R2达到0.997,pKa为2.24。
实施例8
选取实施例3制备的化合物C按照实施例4的方法进行性能测试。如图5所示,化合物C的最大发射波长为515nm,荧光强度随着pH值降低而下降并呈现红移趋势。通过比较pH1.0和pH 6.0条件下515nm处的荧光发射强度,我们发现化合物C的荧光发射强度增加了10倍以上。
实施例9
选取实施例3制备的化合物C进行对pH与荧光强度关系图中点的S型拟合。如图6所示,截取pH 3.0至pH 5.6范围内pH与荧光强度关系图进行线性拟合,化合物C的荧光与pH变化呈良好的线性关系,R2达到0.998,pKa为4.45。
实施例10
选取实施例3制备的化合物C进行选择性测试。干扰离子或分子使用0.1mM的CaCl2、CuCl2、FeCl2、FeCl3、KI、Na2SO4、ZnCl2、Cys、Hys、GSH、H2O2、VC测试前加入待测液并充分混匀。如图7所示,在生理浓度下这些离子或分子对化合物C的荧光强度没有显著影响。
实施例11
选取实施例3制备的化合物C进行荧光可逆性测试。如图8所示,化合物C可以可逆地进行至少五个周期,这表明其能够实时有效监测pH波动。
实施例12
选取实施例3制备的化合物C进行细胞成像测试。HeLa细胞使用添加10%热灭活胎牛血清、1%抗生素的完全DMEM培养基,培养条件为恒温37℃、5%CO2,细胞经过胰酶消化处理后,将Hela细胞接种于35mm培养皿中,每孔约5×104个细胞,450μL完全培养基。培养24小时之后,加入浓度为10μM的化合物C,药物处理24小时后吸弃培养基,用PBS洗3次,然后分别加入pH 1.0、2.0、3.0、4.0、5.0、6.0的柠檬酸-柠檬酸钠缓冲液温育30分钟,后置于激光共聚焦扫描显微镜下成像。激发波长为405nm,接受波长为500–520nm。如图9所示,化合物C进入了细胞并在激光照射下产生蓝色的荧光,随着pH的升高荧光强度逐渐增强。这一结果表明化合物C能有效分辨细胞内环境的pH变化。
Claims (8)
2.权利要求1所述的吲哚菁类衍生物的制备方法,其特征在于,包括如下步骤:依次向60mL乙醇中2,3,3-三甲基-4,5-苯并吲哚、甲醛类化合物和哌啶进行反应,然后将反应溶液加热并在氮气气氛下回流,冷却至室温后减压去除溶剂,并通过快速柱色谱法获得产物。
3.根据权利要求2所述的吲哚菁类衍生物的制备方法,其特征在于,所述的2,3,3-三甲基-4,5-苯并吲哚、甲醛类化合物和哌啶的添加量分别为10mmol,12mmol及0.1mmol。
4.根据权利要求2所述的吲哚菁类衍生物的制备方法,其特征在于,所述的加热是将反应溶液升温到120℃。
5.根据权利要求2所述的吲哚菁类衍生物的制备方法,其特征在于,所述回流的时间为18h。
6.根据权利要求2所述的吲哚菁类衍生物的制备方法,其特征在于,所述的甲醛类化合物是3-氰基苯甲醛、3-羧基苯甲醛及香豆素-6-甲醛之一。
7.权利要求1所述的吲哚菁类衍生物在pH荧光探针上的应用。
8.根据权利要求7所述的吲哚菁类衍生物在pH荧光探针上的应用,其特征在于,所述pH的检测范围为1.0-7.0。
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