摘要
目的:分析合成的n=20-mer硫代寡核苷酸中相关物质“n-1”、“ n-2”和“ n-3”杂质失败序列。方法:离子交换高效液相色谱(IE-HPLC)法和聚丙烯酰胺凝胶电泳法(PAGE)。色谱柱为Gen-Pak^(TM)FAX离子交换柱(4.6 mm×100mm),流动相A为62.5mmol·L^(-1)三羟甲基氨基甲烷盐酸(Tris·Cl),pH 8.15,流动相B为62.5mmol·L^(-1) Tris·Cl,pH 8.15,2.5 mol·L^(-1) LiCl,流动相C为100％乙腈;梯度洗脱条件为B:30％→50％ 30 min,C:恒为20％;流速为0.75mL·min^(-1);检测波长为260nm。PAGE分析采用20％变性聚丙烯酰胺凝胶,恒定功率25W进行电泳。结果:IE-HPLC分离纯化出3种相关物质,通过PAGE印证了它们是由于合成偶联不完全而产生的n-1、n-2和n-3的杂质失败序列。结论:所用离子交换高效液相色谱法能将合成的硫代寡核苷酸中相关物质“n-1”、“n-2”和“n-3”杂质失败序列与全序列n一一分离出来,对硫代寡核苷酸的纯化和分析具有重要的参考价值。
Objective: To analyse the relative substances for the synthesized phosphorothioate oligonucleotides of n = 20 - mer. Method: Ion exchange HPLC (IE - HPLC) and PAGE. The ion - exchange column was Gen -PakTMFAX (4. 6 mm × 100 mm). The mobile phase A was 62. 5 mmol L-1 Tris· Cl, pH 8. 15 , and the mobile phase B was 62. 5 mmol· L-1 Tris ?Cl, 2. 5 mol · L-1 LiCl, pH 8. 15, and the mobile phase C was 100% aceto-nitrile. The condition of gradient elution was B: 30% →50% 30 min, and C : 20% . The flow rate was 0. 75 mL· min-1 . The detection was done at 260 nm. PAGE condition were 20% polyacrylamide and constant power at 25 W to electrophoresize. Result: Three kinds of relative substance from the synthetic phosphorothioate oligonucleotides were separated by using ion - exchange HPLC. On the basis of the PAGE method, it was confirmed that they were the failure sequences of n - 1 , n - 2 and n - 3 which were produced from the incomplete coupling when synthesized. Conclusion: The relative substances n - 1 , n -2 and n -3 for the synthesized phosphorothioate oligonucleotides can be separated one by one by using the ion - exchange HPLC analysis method, which has reference value for the purification and the analysis of phosphorothioate oligonucleotides.
出处
《药物分析杂志》
CAS
CSCD
北大核心
2002年第5期371-375,共5页
Chinese Journal of Pharmaceutical Analysis
