双功能试剂与螯合金属元素标记多肽新技术的建立

New Peptide Labeling Technique Based on Bifunctional Reagent and Rare Earth Metal Chelating Tags

建立了双功能试剂2-[(4-异硫氰基苯基)甲基]-1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸(p-SCN-Bn-DOTA)与标准肽段反应的新方法,测定了反应产物与镧系金属Eu(Ⅲ)的螯合效率,探索了其作为靶向探针和蛋白定量标记试剂性能。考察了不同缓冲体系的浓度及pH值、p-SCN-Bn-DOTA量与肽段相对量、反应体系中有机相与水相比例,反应温度与反应时间对偶联效率的影响。实验结果显示:p-SCN-Bn-DOTA量为肽段的32倍,缓冲体系为0.2 mol/L TEAB(pH 8.5),60℃反应60 min,有机相与水相之比为4.4∶1(V/V)时标记效率高;在HCl/Na Ac缓冲体系(pH 4.0)中60℃反应60 min,金属离子鳌合反应效率高达94%。本研究建立了一种新的基于双功能试剂p-SCN-Bn-DOTA的标记方法,并成功用于RGD肽等的标记。

A novel labeling approach with good compatibility based on 2-（4-isothiocyanatobenzyl）-1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid （p-SCN-Bn-DOTA） was established. The labeling conditions of p-SCN-Bn-DOTA with standard peptides were optimized, and the labeling efficiency of macrocyclic complex of p-SCN-Bn-DOTA with lanthanide metal ion Eu（Ⅲ） was also investigated. The labeling process was a two-step reaction. The labeling efficiency was affected by several factors including pH, buffer, reaction time, reaction temperature, and ratio of p-SCN-Bn-DOTA to peptide. The optimized conditions were as followed： the first step of labeling reaction was reacted in 0.2 mol/L triethylene diamine （TEAB） （ pH 8.5 ） buffer for 60 rain at 60℃, and the molar ratio of p-SCN-Bn-DOTA with peptide was 32. The second step of rare earth metal chelating was in pH 4.0 HCI/NaAC buffer for 60 min at 60℃. This approach was successfully applied in RGD peptide labeling.