A novel chiral thiol reagent for automated precolumn derivatization and high-performance liquid chromatographic enantioseparation of amino acids and its application to the aspartate racemase assay.
A novel chiral thiol reagent for automated precolumn derivatization and high-performance liquid chromatographic enantioseparation of amino acids and its application to the aspartate racemase assay.
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DOI：
10.1016/s0003-2697(02)00705-4
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发表时间：
2003-04
影响因子：
2.9
通讯作者：
N. Nimura;T. Fujiwara;Aya Watanabe;M. Sekine;T. Furuchi;M. Yohda;A. Yamagishi;T. Oshima;H. Homma
中科院分区：
文献类型：
--
作者：
N. Nimura;T. Fujiwara;Aya Watanabe;M. Sekine;T. Furuchi;M. Yohda;A. Yamagishi;T. Oshima;H. Homma
合成了一种新型光学活性硫醇化合物 N-(叔丁基硫代氨基甲酰基)-L-半胱氨酸乙酯 (BTCC) 作为手性衍生试剂。该化合物和邻苯二甲醛与氨基酸对映体反应生成荧光非对映体，可通过 HPLC 在反相柱上轻松分离。使用 BTCC 可以显着改善酸性氨基酸的对映体分离。在本研究中，将新型化合物的对映体分离 HPLC 方法应用于天冬氨酸 (Asp) 消旋酶测定。衍生化的 d-Asp 在 l-Asp 衍生物之前被洗脱。因此，即使在活性非常低的情况下，也可以准确地定量由消旋酶对大量 l-Asp 底物的活性产生的少量 d-Asp。由于衍生化反应在室温下快速进行，因此建立了全自动的衍生化和样品注射系统。该自动化方法实用并成功应用于古菌天冬氨酸消旋酶测定。我们推测该程序还适用于其他氨基酸、氨基醇和儿茶酚胺的对映体分离。
A novel optically active thiol compound, N-(tert-butylthiocarbamoyl)-l-cysteine ethyl ester (BTCC), is synthesized as a chiral derivatization reagent. This compound and o-phthalaldehyde react with amino acid enantiomers to produce fluorescent diastereomers that are readily separable on a reverse-phase column by HPLC. Enantioseparation of acidic amino acids in particular is markedly improved using BTCC. In this study, the HPLC method for enantioseparation with the novel compound is applied to the aspartate (Asp) racemase assay. Derivatized d-Asp is eluted before the l-Asp derivative. Consequently, a small amount of d-Asp produced by the activity of racemase on a large quantity of l-Asp substrate may be quantified accurately, even at very low activity. Since the derivatization reaction proceeds rapidly at room temperature, a fully automated system is established for derivatization and sample injection. The automated method is practical and successfully applied to the archaeal Asp racemase assay. We presume that the procedure is additionally applicable to the enantioseparation of other amino acids, amino alcohols, and catecholamines.