薄层色谱法纯化卟啉锰修饰的TFO

Purification of manganese porphyrin-TFO with thin layer chromatography

目的 筛选分离纯化卟啉锰 TFO 吖啶化合物的简单有效的方法。方法 以固相亚磷酰胺法合成吖啶、6碳氨基连接臂修饰的TFO ,并以温和法切割和脱保护 ;四苯基卟啉四羧酸经金属化、酯化后 ,与上述TFO偶联 ;分别以变性聚丙酰胺凝胶电泳、寡核苷酸纯化柱、薄层色谱法分离纯化偶联反应产物。结果 以凝胶电泳分离偶联反应产物时 ,偶联反应产物带、卟啉锰活性酯带位于同一水平 ,未分离出独立的卟啉锰 TFO 吖啶化合物带。以寡核苷酸纯化柱分离偶联反应产物时 ,紫外分光光度仪检测结果显示 ,回收物仍为卟啉锰 TFO 吖啶化合物与游离的卟啉锰活性酯的混合物。以薄层层析分离偶联反应产物时 ,在卟啉锰活性单酯带后 ,出现一条绿色月牙型带 ,紫外光光谱分析显示 ,此带回收物同时具有寡核苷酸和卟啉锰的特征吸收峰 (UVλmax =2 6 0、4 6 8nm) ,质谱分析结果证实 ,回收物分子量实测值与卟啉锰 TFO 吖啶化合物理论一致。结论 薄层色谱法可简单有效分离纯化卟啉锰 TFO 吖啶化合物。

Objective To filtrate simple effective method separating and purifying manganese porphyrin triple helix forming oligonucleotide (TFO) acridine compounds. Method TFO modified by acridine and amino modifier C6 were synthesized by solid phase phosphoramidite chemistry, then deprotected mildly. Meso tetrakis(4 carbocyphenyl) porphyrin was prepared by metallation and esterification, then linked with previously described oligonucleotides. The reaction products were separated and purified by denaturing polyacrylamide gel, oligonucleotide purification cartridge and thin layer plate respectively. Results When the reaction products were separated by denaturing polyacrylamide gel, the reaction products strand and manganese porphyrin activated esters strand were at same level, manganese porphyrin TFO acridine compounds strand was not observed. After the reaction products were separated by oligonucleotide purification cartridge, the products were still mixed of manganese porphyrin TFO acridine compounds and manganese porphyrin activated esters, which was made sure by ultraviolet absorption spectrometry. When the reaction products were separated by thin layer plate, there was a green lunarform strand behind manganese porphyrin monoactivated esters, the reclaim of this strand possess characteristic ultraviolet absorption peak of oligonucleotide and manganese porphyrin, which was show by ultraviolet absorption spectra. Mass spectrogram confirmed that the observed mass of TFO derivative were same as that of the theoretical mass. Conclusion Manganese porphyrin TFO acridine compounds were separated and purified simply and effectively by thin layer plate.