CN113979981A - 一种巯基响应型脱碱基位点捕获试剂及应用 - Google Patents
一种巯基响应型脱碱基位点捕获试剂及应用 Download PDFInfo
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- CN113979981A CN113979981A CN202111232481.5A CN202111232481A CN113979981A CN 113979981 A CN113979981 A CN 113979981A CN 202111232481 A CN202111232481 A CN 202111232481A CN 113979981 A CN113979981 A CN 113979981A
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Abstract
本发明公开一种巯基响应型脱碱基位点捕获试剂及应用,AP捕获试剂选自2,4‑二硝基苯磺酰化的烷氧基胺或2,3,4,5,6‑五氟苯磺酰化的烷氧基胺;AP捕获试剂在含有巯基化合物的激活作用下生成烷氧基胺,烷氧基胺与AP位点反应生成肟,破坏DNA分子结构,使其不能复制,最终导致细胞的死亡。本发明利用癌细胞中存在高浓度的谷胱甘肽对AP捕获试剂靶向引导,激活后形成的烷氧基胺与癌细胞中大量存在的AP位点反应,达到杀死癌细胞的目的。AP捕获试剂具有极高靶向性,同时具有突出的细胞毒性和光细胞毒性。
Description
技术领域
本发明涉及一种巯基响应型脱碱基位点捕获试剂及应用。
背景技术
肿瘤细胞是由基因突变引发产生的,其在体内的不可控增长,严重危害人类的健康和生命。手术切除、放疗、化疗、免疫治疗和靶向治疗的联合应用可以有效地提高肿瘤的治愈率和病人的生活质量。在癌症治疗中被广泛使用的DNA抑制剂或烷基化试剂可以引发DNA损伤而导致肿瘤细胞凋亡。抗癌化疗药物在杀伤肿瘤细胞的同时,对机体正常细胞,特别是增殖旺盛的细胞具有损害作用,从而引发一些毒副作用,如:恶心、呕吐、脱发、白细胞减少等。如何增强抗癌药物对肿瘤细胞的靶向性,减少对正常细胞的毒副作用,是抗肿瘤化疗药物研究的热点和难点。
寻找新的作用靶点和靶向激活方法是靶向抗癌化疗药物研发的一种重要策略。脱碱基位点是一种常见的DNA损伤,源于N-糖苷键断裂而使碱基脱落。辐射、烷基化试剂和一些抗癌药物等可能会造成碱基脱落,因此脱碱基位点作为标志性损伤能够帮助疾病早期筛查、药物毒副作用评价、环境污染物毒性评价等。另一方面,由于脱碱基位点产生于正常碱基的缺失脱落,因此在双链DNANc双螺旋结构中形成一个空腔,允许小分子进入其中,空腔的体积对进入的小分子具有一定的空间选择性。由于空腔位于双链核酸的内部,具有疏水特性。另外,脱碱基位点对面的碱基,由于失去了配对的碱基,重新具备了形成氢键的能力。因此,结构合适的小分子,能够在空腔内通过疏水和氢键作用,与核酸分子形成稳定的复合物。脱碱基位点的以上特性,使之被应用于小分子检测、核苷酸/SNP检测以及适配体生物传感器构建等方面的研究工作中。目前已有不同的检测方法用于脱碱基位点的定量、定性分析,包括32P后标记法、LC-MS、ELISA及化学探针检测法等。另一方面,由于脱碱基位点在双链DNA内形成疏水空腔,能够结合小分子,使得脱碱基位点作为结合位点被用于小分子检测、构建适配体传感器及SNP检测。肿瘤细胞中每天会产生大量脱碱基(apurinic/apyrimidinic,AP)位点,但会被及时识别修复,无法诱发肿瘤细胞凋亡。脱碱基位点作为DNA中最重要的损伤标志物,如何利用这一天然的靶标改善和减轻抗癌药物对人体正常细胞的毒副作用,实现潜在靶向抗癌药的研发是一个新的科学问题。
发明内容
为解决上述技术问题,本发明提供一种巯基响应型脱碱基位点捕获试剂、制备方法及应用。
本发明采用的技术方案是:一种化合物,结构如式1所示:
其中R1为甲基、乙基、丙基、异丙基、丙炔基、丁基、异丁基、苯基、7-乙氧基香豆素、7-乙氧基-4-甲基香豆素、7-(二乙胺基)香豆素或7-(二乙胺基)-4-甲基香豆素。
一种化合物,结构如式2所示:
其中R2为甲基、乙基、丙基、异丙基、丙炔基、丁基、异丁基、苯基、7-乙氧基香豆素、7-乙氧基-4-甲基香豆素、7-(二乙胺基)香豆素或7-(二乙胺基)-4-甲基香豆素。
一种巯基响应型脱碱基位点捕获试剂,包括式1化合物或式2化合物。
优选地,式1化合物或式2化合物与含有巯基化合物反应,生成含有烷氧基的化合物,烷氧基能够与脱碱基位点反应。
优选地,含有巯基化合物为谷胱甘肽或巯基乙醇。
优选地,式1化合物或式2化合物与癌细胞中高浓度谷胱甘肽反应。
优选地,式1化合物或式2化合物中包含香豆素单元,用于捕获试剂时置于紫外光照条件下。
巯基响应型脱碱基位点捕获试剂在制备靶向抗癌药物中的应用。
巯基响应型脱碱基位点捕获试剂在制备肿瘤筛查制剂中的应用,式1化合物或式2化合物中包含香豆素单元。
本发明具有的优点和积极效果是:捕获试剂能够与含有巯基化合物反应,生成含有烷氧基的化合物,烷氧基能够与脱碱基位点反应生成肟,从而导致细胞的死亡,由于肿瘤细胞中含有高浓度的谷胱甘肽,捕获试剂能够靶向定位到肿瘤细胞处,在谷胱甘肽的激活作用下,对肿瘤细胞产生影响,促进肿瘤细胞的死亡;
捕获试剂具有靶向性,含有香豆素单元的捕获试剂还能够用于肿瘤早期检测;尤其当其与紫外光联用时,能够产生DNA链间交联产物,进一步促进肿瘤细胞的死亡,具有极高靶向性,同时具有突出的细胞毒性和光细胞毒性。
附图说明
图1为实施例7中式3化合物与巯基乙醇(BME)反应,荧光增强图谱;
图2为实施例7中式4化合物与巯基乙醇(BME)反应,荧光增强图谱;
图3为实施例7中式7化合物与巯基乙醇(BME)反应,荧光增强图谱;
图4为实施例8中式3化合物与谷胱甘肽(GSH)反应,荧光增强图谱;
图5为实施例8中式4化合物与谷胱甘肽(GSH)反应,荧光增强图谱;
图6为实施例8中式7化合物与谷胱甘肽(GSH)反应,荧光增强图谱;
图7为实施例9中AP捕获试剂在肾正常细胞与肾癌细胞的激光共聚焦显微镜成像;
图8为实施例10中AP捕获试剂在肾正常细胞与肺癌细胞的激光共聚焦显微镜成像;
图9为实施例11中AP捕获试剂在肾正常细胞与肺癌细胞的激光共聚焦显微镜成像;
图10为实施例12细胞周期分析图;
图11为实施例13中甲氧基胺抑制APE1酶修复AP位点;
图12为实施例13中APE1酶无法修复被捕获的AP位点。
具体实施方式
本发明设计巯基响应型脱碱基位点捕获试剂,包括式1化合物或式2化合物。捕获试剂选自2,4-二硝基苯磺酰化的烷氧基胺或2,3,4,5,6-五氟苯磺酰化的烷氧基胺。
其中,2,4-二硝基苯磺酰化的烷氧基胺结构如式1所示:
其中R1为甲基、乙基、丙基、异丙基、丙炔基、丁基、异丁基、苯基、7-乙氧基香豆素、7-乙氧基-4-甲基香豆素、7-(二乙胺基)香豆素或7-(二乙胺基)-4-甲基香豆素;
其中,2,3,4,5,6-五氟苯磺酰化的烷氧基胺结构如式2所示:
其中R2为甲基、乙基、丙基、异丙基、丙炔基、丁基、异丁基、苯基、7-乙氧基香豆素、7-乙氧基-4-甲基香豆素、7-(二乙胺基)香豆素或7-(二乙胺基)-4-甲基香豆素;
AP捕获试剂能够与含有巯基化合物反应,生成二氧化硫和含有烷氧基的化合物,烷氧基能够与脱碱基位点反应生成肟,从而导致细胞死亡。AP捕获试剂能够与谷胱甘肽或巯基乙醇中的巯基反应,本发明某些实施例中,可利用癌细胞中大量谷胱甘肽激活AP捕获试剂,释放烷氧基,与癌细胞中脱碱基位点结合后,诱导癌细胞死亡,从而实现靶向抗癌活性剂光化学抗癌活性的目的。
本发明某些实施例中,当AP捕获试剂中包含香豆素单元时,AP捕获试剂与巯基反应会产生有荧光的烷氧基胺,可通过检测对其强度和位置进行检测,AP捕获试剂则可用于制备肿瘤筛查制剂。另外,当AP捕获试剂中包含香豆素单元时,还可以与紫外光协作,能够产生DN链间交联产物,进一步促进细胞死亡。
下面结合附图对本发明方案做出说明,其中,未具体说明操作步骤的实验方法,均按照相应商品说明书进行,实施例中所用到的仪器、试剂、耗材如无特殊说明,均可从商业公司购买得到。
实施例1:
式3化合物合成路径如下所示:
化合物1的合成:称取邻苯二酚(2.2g,1.0eq)和乙酰乙酸乙酯(2.9g,1.1eq)用甲苯(30mL)溶解,加入磷酸(2g),该混合物回流2小时。反应结束,加水(60mL)淬灭反应,有黄色固体析出,抽滤,滤饼用乙醇洗,得到目标化合物1(2.7g),黄色固体,产率为76%,1H NMR(400MHz,CDCl3)δ7.50(d,J=8.7Hz,1H),6.89(d,J=2.4Hz,1H),6.83(dd,J=8.6,2.5Hz,1H),6.16(s,1H),2.41(s,3H)。
化合物2的合成:称取化合物1(0.3g,1.0eq)用丙酮(10mL)溶解,加入1,2-二溴乙烷(1.3g,4.0eq)和碳酸钾(0.7g,3.0eq)。该混合物在回流条件下反应过夜。反应结束,冷却至室温,旋走溶剂,得到的粗品用硅胶色谱纯化,洗脱剂为乙酸乙酯-石油醚(1:4-1:2),得到化合物2(0.18g),白色固体粉末,产率为27%,1H NMR(400MHz,CDCl3)δ7.45(d,J=8.8Hz,1H),6.83(dd,J=8.8,2.5Hz,1H),6.75(d,J=2.5Hz,1H),6.09(d,J=0.9Hz,1H),4.29(t,J=6.1Hz,2H),3.61(t,J=6.1Hz,2H),2.34(s,3H)。
化合物3的合成:称取化合物2(0.2g,1.0eq)和2-羟基异吲哚啉-1,3-二酮(0.18g,1.6eq)用N,N-二甲基甲酰胺(5mL)溶解,加入碳酸钾(0.3g,3.2eq)。该混合物在室温下搅拌直到反应结束。加水淬灭,用乙酸乙酯萃取,收集有机层,无水硫酸钠干燥,真空浓缩,得到的粗品化合物3(0.1g),黄色油,产率为39%,1H NMR(400MHz,CDCl3)δ7.78(dd,J=5.5,3.1Hz,2H),7.71(dd,J=5.5,3.1Hz,2H),7.42(d,J=8.8Hz,1H),6.75(dd,J=8.7,2.5Hz,1H),6.68(d,J=2.5Hz,1H),6.08(s,1H),4.55(t,J=5.3,3.3Hz,2H),4.35(t,J=5.4,3.4Hz,2H),2.33(s,3H)。
化合物4的合成:称取化合物3(0.12g,1.0eq)用甲醇(8mL)溶解,加入80%水合肼(0.02g,1.0eq)。该混合物在室温下搅拌2小时。反应结束以后,旋走溶剂,得到的残渣用乙酸乙酯(15mL)和水(20mL)萃取,分离有机层,水层用乙酸乙酯反萃(10mL×3),合并有机层,无水硫酸钠干燥,真空浓缩,得到的粗品用硅胶色谱纯化,洗脱剂为乙酸乙酯-石油醚(1:4-1:1),得到化合物4(0.02g),白色固体粉末,产率为30%,1H NMR(400MHz,DMSO)δ7.74(t,J=7.9Hz,1H),7.04(dt,J=8.7,2.3Hz,2H),6.28(t,J=3.9Hz,1H),6.20(s,2H),4.28(t,2H),3.92(t,2H),2.45(s,3H)。
化合物5的合成:称取化合物4(0.06g,1.0eq)用重蒸的吡啶(1mL)溶解,在氩气保护条件下,进行无水无氧操作三次,再加入2,4-二硝基苯磺酰氯(0.2g,3.2eq)。该混合物在室温下搅拌2小时,反应结束,旋走吡啶,得到的粗品用乙酸乙酯(10mL)溶解,加水(20mL)萃取,分离有机层,水层用乙酸乙酯反萃(5mL×3),合并有机层,无水硫酸钠干燥,真空浓缩,得到的粗品用硅胶色谱纯化,洗脱剂为乙酸乙酯-石油醚(1:2-1:1),得到化合物5(0.09g),黄色固体粉末,产率为75%,1H NMR(400MHz,DMSO)δ11.62(s,1H),8.95(d,J=2.2Hz,1H),8.70(dd,J=8.7,2.3Hz,1H),8.29(d,J=8.7Hz,1H),7.66(d,J=8.6Hz,1H),6.95(dt,J=8.7,2.4Hz,2H),6.21(d,J=0.9Hz,1H),4.27(dd,J=11.8,5.1Hz,4H),2.39(s,3H)。
实施例2:
式4化合物合成路径如下所示:
化合物6的合成:称取4-甲基-7-氨基香豆素(0.3g,1.0eq)用N,N-二甲基甲酰胺(10mL)溶解,加入碳酸钾(1.3g,5.0eq)和碘乙烷(0.85g,3.0eq),该混合物在加热80℃条件下反应2小时。反应结束,冷却至室温,加水(20mL)淬灭,用乙酸乙酯(10mL)萃取,合并有机层,水层用乙酸乙酯反萃(5mL×3)。合并有机层,无水硫酸钠干燥,真空浓缩,得到的粗品用硅胶色谱纯化,洗脱剂为乙酸乙酯-石油醚(1:3-1:1),得到化合物6(0.11g),黄色固体粉末,产率为30%,1H NMR(400MHz,CDCl3)δ7.32(d,J=8.6Hz,1H),6.57(d,J=8.3Hz,1H),6.51(s,1H),5.95(d,J=0.8Hz,1H),3.18(q,J=7.2Hz,2H),2.29(d,J=1.0Hz,3H),1.29-1.21(m,3H)。
化合物7的合成:称取化合物5(0.2g,1.0eq)用N,N-二甲基甲酰胺(10mL)溶解,加入碳酸钾(0.4g,3.0eq)和2-溴乙醇(0.25g,2.0eq)。该反应混合物回流24小时,反应结束,冷却至室温。用乙酸乙酯和水萃取,分离有机层,水层用水反萃三次,合并有机层,无水硫酸钠干燥,真空浓缩,得到的粗品用硅胶色谱纯化,洗脱剂为乙酸乙酯-石油醚(1:3-1:1),得到化合物7(0.08g),黄色固体粉末,产率为33%,1H NMR(400MHz,CDCl3)δ7.31(d,J=8.9Hz,1H),6.65(d,J=7.9Hz,1H),6.52(s,1H),5.89(s,1H),3.79(t,J=5.8Hz,2H),3.48(dd,J=10.3,4.5Hz,2H),3.45-3.40(m,2H),2.27(s,3H),1.14(t,J=7.1Hz,3H)。
化合物8的合成:称取化合物7(0.14g,1.0eq)用重蒸四氢呋喃(5mL)溶解,在氩气保护的条件下,进行无水无氧三次,在加入2-羟基异吲哚啉-1,3-二酮(0.11g,1.2eq)和磷酸三苯酯(0.18g,1.2eq),在0℃条件下,缓慢滴加偶氮二甲酸二异丙酯(0.14g,1.2eq)。该混合物室温搅拌过夜。反应结束,旋走溶剂。得到的粗品用硅胶色谱纯化,洗脱剂为乙酸乙酯-石油醚(2:1),得到化合物8(0.15g),黄色固体粉末,产率为75%,1H NMR(400MHz,CDCl3)δ7.77(d,J=4.0Hz,2H),7.70(d,J=3.1Hz,2H),7.35(d,J=8.9Hz,1H),6.64(d,J=8.9Hz,1H),6.49(s,1H),5.91(s,1H),4.33(t,J=5.7Hz,2H),3.75(t,J=5.7Hz,2H),3.52(q,J=7.0Hz,2H),2.27(s,3H),1.19(t,J=6.8Hz,3H)。
化合物9的合成:称取化合物8(0.1g,1.0eq)用甲醇(5mL)溶解,加入80%水合肼(0.02g,1.0eq)。该混合物在室温下搅拌2小时。反应结束以后,旋走溶剂,得到的残渣用乙酸乙酯(15mL)和水(20mL)萃取,分离有机层,水层用乙酸乙酯反萃(10mL×3),合并有机层,无水硫酸钠干燥,真空浓缩,得到的粗品用硅胶色谱纯化,洗脱剂为乙酸乙酯-石油醚(1:4-1:1),得到化合物9(0.06g),白色固体粉末,产率为85%,所得产物为黄色固体粉末,产率为85%,1H NMR(400MHz,DMSO)δ7.49(d,J=9.0Hz,1H),6.73(dd,J=9.0,2.4Hz,1H),6.56(d,J=2.4Hz,1H),6.12(s,2H),5.94(s,1H),3.68(t,J=5.8Hz,2H),3.63-3.53(m,2H),3.52-3.40(m,2H),2.32(s,3H),1.10(t,J=7.0Hz,3H)。
化合物10的合成:称取化合物9(0.12g,1.0eq)用重蒸的吡啶(10mL)溶解,在氩气保护条件下,进行无水无氧操作三次,再加入2,4-二硝基苯磺酰氯(0.37g,3.0eq)。该混合物在室温下搅拌2小时,反应结束,旋走吡啶,得到的粗品用乙酸乙酯(10mL)溶解,加水(20mL)萃取,分离有机层,水层用乙酸乙酯反萃(5mL×3),合并有机层,无水硫酸钠干燥,真空浓缩,得到的粗品用硅胶色谱纯化,洗脱剂为乙酸乙酯-石油醚(1:2-1:1),得到化合物10(0.12g),黄色固体粉末,产率为54%所得产物为黄色固体粉末,产率为54%,1H NMR(400MHz,DMSO)δ11.56(s,1H),8.88(d,J=2.3Hz,1H),8.55(dd,J=8.7,2.3Hz,1H),8.18(d,J=7.7Hz,1H),7.38(d,J=9.0Hz,1H),6.60(dd,J=9.0,2.5Hz,1H),6.42(d,J=2.5Hz,1H),5.92(d,J=1.1Hz,1H),4.11(t,J=5.2Hz,2H),3.58(t,J=5.1Hz,2H),3.40-3.36(m,2H),2.30(s,3H),1.05(t,J=7.0Hz,3H)。
实施例3:
式5化合物合成路径如下所示:
化合物11的合成:称取羟基氨基甲酸叔丁酯(0.5g,1.0eq)用N,N-二甲基甲酰胺(10mL)溶解,加入碳酸钠(3.9g,2.0eq)和3-溴丙炔(1.3g,3.0eq)。该混合物在室温条件下反应过夜。反应结束用水和乙酸乙酯萃取。合并有机层,无水硫酸钠干燥,真空浓缩,得到的粗品用硅胶柱纯化。得到的产物直接投入到下一步反应。
化合物12的合成:将化合物11(0.1g,1.0eq)用二氯甲烷溶解,加入含有1,4-二氧六环的盐酸溶液(4.0M),在0℃条件下缓慢滴加,20分钟左右,有白色固体析出。1H NMR(400MHz,CD3OD)δ4.748(s,2H),3.376(t,J=2.4Hz,1H)。
化合物13的合成:称取化合物12(0.17g,1.0eq)用重蒸的吡啶(10mL)溶解,在氩气保护条件下,进行无水无氧操作三次,再加入2,4-二硝基苯磺酰氯(0.74g,3.0eq)。该混合物在室温下搅拌2小时,反应结束,旋走吡啶,得到的粗品用乙酸乙酯(10mL)溶解,加水(20mL)萃取,分离有机层,水层用乙酸乙酯反萃(5mL×3),合并有机层,无水硫酸钠干燥,真空浓缩,得到的粗品用硅胶色谱纯化,洗脱剂为乙酸乙酯-石油醚(1:2-1:1),得到化合物13(0.12g),黄色固体粉末,产率为54%。所得产物为砖红色的油,产率为54%,1H NMR(400MHz,CDCl3)δ8.65(d,J=2.2Hz,1H),8.55(dd,J=8.6,2.2Hz,1H),8.39(d,J=8.6Hz,1H),8.25(s,1H),4.61(d,J=2.4Hz,2H),2.52(t,J=2.4Hz,1H)。
实施例4:
式6化合物合成路径如下所示:
化合物14的合成:称取甲氧基胺(0.16g,1.0eq)用重蒸的吡啶(10mL)溶解,在氩气保护条件下,进行无水无氧操作三次,再加入2,4-二硝基苯磺酰氯(0.16g,3.0eq)。该混合物在室温下搅拌2小时,反应结束,旋走吡啶,得到的粗品用乙酸乙酯(10mL)溶解,加水(20mL)萃取,分离有机层,水层用乙酸乙酯反萃(5mL×3),合并有机层,无水硫酸钠干燥,真空浓缩,得到的粗品用硅胶色谱纯化,洗脱剂为乙酸乙酯-石油醚(1:2-1:1),得到化合物14(0.17g),黄色固体粉末,产率为54%所得产物为砖红色的油,产率为54%,1H NMR(400MHz,CDCl3)δ8.64(d,J=2.2Hz,1H),8.54(dd,J=8.6,2.2Hz,1H),8.39(d,J=8.6Hz,1H),8.15(s,1H),3.82(s,3H)。
实施例5:
式7化合物合成路径如下所示:
化合物15的合成:称取2-溴乙烷-1-胺溴酸盐(1.0g,1.0eq)用甲醇(60mL)溶解,加入二碳酸二叔丁酯(2.3g,2.0eq)和三乙胺(7mL),该混合物先在60℃条件下加热1小时,然后在室温下再搅拌14小时。旋走溶剂,用二氯甲烷溶解,再用1.0M HCl溶液洗,饱和食盐水洗,合并有机层,无水硫酸钠干燥,真空浓缩得到无色的油状物(1g),产率92%,1HNMR(400MHz,DMSO)δ7.11(s,1H),3.41(d,J=5.6Hz,2H),3.27(d,J=6.3Hz,2H),1.38(s,9H)。
化合物16的合成:称取7-羟基-4-甲基香豆素(0.6g,1.0eq)用丙酮溶解,加入碳酸钾(0.51g,2.1eq)先回流1小时。然后化合物15(0.83g,2.1eq)。该混合物再继续回流18小时。反应结束,直接旋走溶剂,得到的粗品过硅胶柱纯化。得到化合物16(0.8g),产率80%,1H NMR(400MHz,CDCl3)δ7.43(d,J=8.8Hz,1H),6.79(dd,J=8.8,2.5Hz,1H),6.73(d,J=2.4Hz,1H),6.07(s,1H),4.96(s,1H),4.01(t,J=5.1Hz,2H),3.50(dd,J=9.9,5.1Hz,2H),2.33(s,3H),1.37(d,J=8.9Hz,9H)。
化合物17的合成:称取化合物16(0.1g,1.0eq)用二氯甲烷溶解,缓慢加入三氟乙酸(1.7mL),在室温下反应2小时,反应结束以后,用水淬灭,水层用二氯甲烷反萃三次,合并有机层,无水硫酸钠干燥,真空浓缩得到化合物3(0.05g),产率76%,1H NMR(400MHz,CDCl3)δ7.43(d,J=8.8Hz,1H),6.81(dd,J=8.8,2.5Hz,1H),6.76(d,J=2.4Hz,1H),6.08(d,J=0.8Hz,1H),3.98(t,J=5.1Hz,2H),3.07(t,J=5.1Hz,2H),2.33(s,3H)。
化合物18的合成:称取化合物17(0.1g,1.0eq)用重蒸的吡啶溶解,进行无水无氧操作三次,再加入2,3,4,5,6-五氟苯-1-磺酰氯(0.37g,3.0eq)。该混合物在室温下搅拌3小时。反应结束,旋走溶剂,得到的粗品过硅胶柱纯化,得到化合物4(0.1g),产率62%,1H NMR(400MHz,CDCl3)δ7.43(d,J=8.7Hz,1H),7.20(d,J=1.4Hz,1H),6.73–6.63(m,1H),6.10(s,1H),5.76(s,1H),4.08(t,J=4.4Hz,2H),3.59(d,J=3.9Hz,2H),2.34(s,3H)。
实施例6:
式8化合物合成路径如下所示:
化合物19的合成:称取甲氧基胺(0.1g,1.0eq)用重蒸的吡啶溶解,进行无水无氧操作三次,再加入2,3,4,5,6-五氟苯-1-磺酰氯(0.37g,3.0eq)。该混合物在室温下搅拌3小时。反应结束,旋走溶剂,得到的粗品过硅胶柱纯化,得到化合物19(0.1g),产率52%,1HNMR(400MHz,CDCl3)δ7.43(d,J=8.7Hz,1H),7.20(d,J=1.4Hz,1H),6.73–6.63(m,1H),6.10(s,1H),4.08(t,3H)。
实施例1-6中公开了部分式1化合物或式2化合物具体合成方法,其他未具体公开其合成方法的AP捕获试剂具有相似的合成路线,或可在现有公开的技术方案基础上,以相同或相似的方法进行合成,本领域技术人员可根据其具体结构特征设计并合成得到相应的化合物。
为了进一步验证AP捕获试剂的作用,选择式3化合物、式4化合物和式7化合物进行细胞实验。
实施例7:AP捕获试剂与巯基乙醇反应
分别取式3化合物、式4化合物和式7化合物与巯基乙醇进行反应,反应路径和试验方法如下。
合成的式3化合物溶解在含有10%DMSO,pH=7.2的缓冲溶液中,试剂的最终浓度为50uM,巯基乙醇(BME)浓度为25mM,式3化合物的激发波长为320nm,发射波长为380nm,反应1小时测荧光,如图1所示,可以明显观察到荧光增强,荧光增强26倍。
合成的式4化合物溶解在含有10%DMSO,pH=7.2的缓冲溶液中,试剂的最终浓度为50uM,巯基乙醇(BME)浓度为25mM,式4化合物的激发波长为380nm,发射波长为460nm,反应1小时测荧光,如图2所示,可以明显观察到荧光增强,荧光增强23倍。
合成的式7化合物溶解在含有10%DMSO,pH=7.2的缓冲溶液中,试剂的最终浓度为50uM,巯基乙醇(BME)浓度为25mM,式7化合物的激发波长为380nm,发射波长为460nm,反应1小时测荧光,如图3所示,可以观察到荧光增强明显。
实施例8:AP捕获试剂与谷胱甘肽反应
分别取式3化合物、式4化合物和式7化合物与巯基乙醇进行反应,反应路径和试验方法如下。
合成的式3化合物溶解在含有10%DMSO,pH=7.2的缓冲溶液中,试剂的最终浓度为50uM,谷胱甘肽(GSH)浓度为25mM,式3化合物的激发波长为320nm,发射波长为380nm,反应1小时测荧光,如图4所示,可以明显观察到荧光增强,荧光增强31倍。
合成的式4化合物溶解在含有10%DMSO,pH=7.2的缓冲溶液中,试剂的最终浓度为50uM,谷胱甘肽(GSH)浓度为25mM,式4化合物的激发波长为380nm,发射波长为460nm,反应1小时测荧光,如图5所示,可以明显观察到荧光增强,荧光增强27倍。
合成的式7化合物溶解在含有10%DMSO,pH=7.2的缓冲溶液中,试剂的最终浓度为50uM,谷胱甘肽(GSH)浓度为25mM,式7化合物的激发波长为380nm,发射波长为460nm,反应1小时测荧光,如图6所示,可以观察到荧光增强不是很明显。所以在后面的研究中没有选择式7化合物。
实施例9:AP捕获试剂用于肾癌细胞实验
本实施例中,选用式4化合物作为AP捕获试剂,将式4化合物(10uM)与肾癌细胞A7860和正常人肾上皮细胞293T在37℃条件下分别孵育1小时,检测前用缓冲液洗2次。通过激光共聚焦显微镜我们发现A7860肾癌细胞的荧光强度是正常肾上皮细胞293T细胞的2.2倍,表明A7860肾癌细胞中GSH的含量高于293T正常肾细胞,所以能够把式4化合物激活,使2,4-二硝基苯磺酰基离去,所以导致荧光增强,如图7所示。
实施例10:AP捕获试剂用于肺癌细胞实验
选用式4化合物作为AP捕获试剂,将式4化合物(10uM)与肺癌细胞H1299和肺正常细胞WI38在37℃条件下分别孵育1小时,检测前用缓冲液洗2次。通过激光共聚焦显微镜我们发现H1299肺癌细胞的荧光强度是肺正常细胞WI38的2.2倍,表明H1299肺癌细胞中GSH的含量高于WI38正常细胞,所以能够把式4化合物激活,使2,4-二硝基苯磺酰基离去,所以导致荧光增强,如图8所示。
实施例11:AP捕获试剂用于肺癌细胞实验
选用式7化合物作为AP捕获试剂,将式4化合物(10uM)与肺癌细胞H1299和肺正常细胞WI38在37℃条件下分别孵育1小时,检测前用缓冲液洗2次。通过激光共聚焦显微镜我们发现H1299肺癌细胞的荧光强度是肺正常细胞WI38的2.2倍,表明H1299肺癌细胞中GSH的含量高于WI38正常细胞,所以能够把式7化合物激活,使2,4-二硝基苯磺酰基离去,所以导致荧光增强,如图9所示。
由实施例9-11能够看出,AP捕获试剂能够靶向作用于癌细胞,在癌细胞中的GSH的激活作用下,释放出具有强荧光的烷氧基胺。但是通过激光共聚焦结果来看,含有2,4二硝基结构的捕获试剂要比2,3,4,5,6-氟取代的捕获试剂荧光增强更明显。
实施例12:细胞凋亡实验
以式3化合物为AP捕获试剂,进行细胞凋亡实验分析。
将H1299肺癌细胞接种于6cm大小的微孔板中,在37℃培养至80-90%融合。用AP捕获试剂(10μM,0.5%DMSO)在37℃下处理细胞24h。在0.25%胰蛋白酶EDTA溶液的帮助下收集细胞。通过1000rpm离心5分钟,将细胞从溶液中取出,并用冰冷的PBS缓冲液洗涤两次。将细胞重新悬浮在500μl结合缓冲液中,并在室温下用5μl Annexin V-FITC和5μl碘化丙啶(PI)溶液染色10分钟,然后通过流式细胞术进行分析。
将H1299肺癌细胞接种于6cm大小的微孔板中,在37℃培养至80-90%融合。用AP捕获试剂(5μM,0.5%二甲基亚砜)在37℃下处理细胞(350nm,1h),并用0.25%胰蛋白酶EDTA溶液收集。以1000rpm的转速离心5分钟,将细胞从溶液中取出,用冰冷的PBS缓冲液洗涤两次,然后用70%冷冻乙醇固定过夜。在用PBS缓冲液洗涤两次后,用碘化丙啶溶液(含0.1%Triton X-100、0.1mmol/L EDTA和50mg/mL RNase A的PBS中的50mg/mL缓冲液)在4℃下在黑暗中染色细胞DNA 30min。在Accuri上分析PI-DNA复合物TMC6 FACScan流式细胞仪(BDBiosciences)和BD CFlow软件v.264.15(BD Biosciences),以获得不同阶段(G1、S和G2/M)的细胞百分比。
通过多参数流式细胞术分析AP捕获试剂对H1299肺癌细胞的细胞反应。如图10所示,结果表明,AP捕获试剂和“AP捕获试剂+光解”的处理均能显著促进早期细胞凋亡。早期凋亡的细胞百分比,未经处理的细胞的早期凋亡的细胞百分比为4%,经AP捕获试剂处理的细胞的早期凋亡的细胞百分比为41%,AP捕获试剂处理联合光解处理的细胞,其早期凋亡的细胞百分比进一步增加到60%。此外,大多数抗肿瘤药物通常以DNA为靶点,以抑制DNA复制,从而使细胞周期停滞在S或G2/M期。细胞周期分析显示,细胞生长停滞在G2/M期。总的来说,AP捕获试剂诱导早期细胞凋亡和G2/M期阻滞,并显示细胞毒性和光细胞毒性。
实施例13:
不含有香豆素单元的AP捕获试剂,不能通过荧光检测判断其效果。本实施例在分子水平通过验证烷氧基胺能够与AP位点结合,APE1酶无法对DNA进行修复,所以DNA序列会被切断,所以会导致DNA无法进行复制与合成,从而验证AP捕获试剂对诱导癌细胞死亡的作用。以式6化合物作为AP捕获试剂。
先利用DNA固相合成仪合成目标ODN 1序列
ODN 1序列::5’-FAM-d(AGC GAC GCT AGU CAA CAC CAA);
利用PAGE法分离纯化得到纯的DNA序列,配制成25uM溶液,进行后续的实验。
首先用UNG酶(尿嘧啶-N-糖基化酶,含ODN的20pmol dU的1单位)处理ODN 1序列,放置在37℃水浴锅中1小时,使得ODN 1序列生成AP位点;再分别加入APE 1酶和试剂3再在室温下孵育2小时,通过PAGE胶分析实验结果。
序列ODN 1生成AP位点以后,在没有加入甲氧基胺的情况下,APE 1酶可以对ODN 1序列进行修复,并将其进行酶切;但是,当加入AP捕获试剂以后,烷氧胺基可以对AP位点进行捕获,这样APE 1酶就不能对其修复,无法进行酶切。实验结果如图11所示,电泳图中从左到右依次为未加AP捕获试剂和APE 1酶、未加AP捕获试剂加入APE 1酶、加入了AP捕获试剂未加APE 1酶和加入了AP捕获试剂和APE 1酶的样品。能够看出,只加了APE 1酶的样品得到了酶切后的序列,同时加入AP捕获试剂和APE 1酶的样品中并未得到酶切后的序列;验证了AP捕获试剂对含有AP位点的DNA序列的破坏。另外如图12所示,只有ODN 1序列生成AP位点以后,烷氧胺基才能对AP位点捕获,捕获以后APE 1酶无法修复,这样也就阻止了DNA的复制。
以上对本发明的实施例进行了详细说明,但所述内容仅为本发明的较佳实施例,不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等,均应仍归属于本发明的专利涵盖范围之内。
Claims (9)
3.一种巯基响应型脱碱基位点捕获试剂,其特征在于:包括权利要求1或权利要求2所述化合物。
4.根据权利要求3所述的巯基响应型脱碱基位点捕获试剂,其特征在于:式1化合物或式2化合物与含有巯基化合物反应,生成含有烷氧基的化合物,烷氧基能够与脱碱基位点反应。
5.根据权利要求4所述的巯基响应型脱碱基位点捕获试剂,其特征在于:所述含有巯基化合物为谷胱甘肽或巯基乙醇。
6.根据权利要求5所述的巯基响应型脱碱基位点捕获试剂,其特征在于:式1化合物或式2化合物与癌细胞中高浓度谷胱甘肽反应。
7.根据权利要求3-6中任一所述的巯基响应型脱碱基位点捕获试剂,其特征在于:式1化合物或式2化合物中包含香豆素单元,用于捕获试剂时置于紫外光照条件下。
8.权利要求3-7中任一所述的巯基响应型脱碱基位点捕获试剂在制备靶向抗癌药物中的应用。
9.权利要求3-7中任一所述的巯基响应型脱碱基位点捕获试剂在制备肿瘤筛查制剂中的应用,其特征在于:式1化合物或式2化合物中包含香豆素单元。
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