合成Hg-DADPA柱分离含硒或硫化合物的研究

Synthesis of Hg-DADPA Column and Separating Selenium or Sulfur Compounds

[目的]通过化学方法合成一种新颖的Hg-DADPA柱,用于分离和富集植物及微生物体中硫硒修饰的化合物。[方法]使用二氨基二异丙基琼脂糖(DADPA)与对氯汞苯甲酸进行反应,得到Hg-DADPA色谱柱,用不同浓度的盐酸洗脱不同位置取代的硫或硒化合物;然后,通过Hg-DADPA色谱富集大肠杆菌(E.coli)的tRNA中含硫、含硒化合物,并且鉴定。同时,测试Hg-DADPA色谱柱分离后样品回收率。[结果]合成的Hg-DADPA柱通过0.002、0.005、0.5 mol/L HCl洗脱,可以分离尿苷三磷酸(UTP)、2-硫代尿嘧啶(2-SU)、2-硒代尿嘧啶(2-Se U)和4-硒代尿苷三磷酸(4-Se UTP)混合物,回收率均在90%以上。Hg-DADPA柱从大肠杆菌K12中选择性地富集到3个含硫修饰的核苷酸,经ESI和HR-MS鉴定为鸟嘌呤-2-硫代尿嘧啶核苷酸、腺嘌呤-3-硫代尿嘧啶核苷酸和鸟嘌呤-5-甲酰-2-尿嘧啶核苷酸。[结论]合成的Hg-DADPA柱对含硒、含硫化合物有很好的分离效果,且可反复使用,从而为植物、微生物中含硒或硫有机化合物的富集、分离提供一个简便的分离材料。

[ Objective ] A novel Hg-DADPA column was synthesized for separation and enrichment selenium and sulfur derivatives from plants and micro-organisms. [ Method] We used DADPA agarose coupled to p-chloromercuribenzoate by EDC crosslinker with different concentration of HC1 elution of different position substituted sulfur or selenium compounds, and then the enrichment of E. coli tRNA sulfur containing selenium compounds and identified through Hg-DADPA chromatographic. The recovery of samples was also tested by Hg-DADPA column. [ Result] It was available for handy and selective separation of the thiouridines and selenium derivatives from mixture. 2-thiouridine, 2-selenouride and 4-sele- nouridine were selectively separated from their mixtures with major nucleotides by 2, 5 and 0.5 mol/L HC1 elution ,and the recovery rate was a- bove 90%. 2-position was easy to elute than 4-position in all the sulfur and selenium nucleotide derivatives. Oligonucleotides containing 4-thio- uridilic acid from RNase A digests of unfractionated ribonueleic acid of E. coli KI2 could be fractionated by elution from such column with differ- ent conditions of HCI concentration. We identifyed three compounds, such as G-4-thiouride acid, A-4-thiouride acid and G-5-formyl-4-thiouride acid, by UV, ESI-MS and High Resolution Mass. [ Conclusion] Newly-synthesized Hg-DADPA column have good separation effect on sulfur and selenium organic compounds, and can be used repeatedly. It provides a simple parting material for enrichment and separation of sulfur and sele- nium organic compounds in plants and microorganisms.