Enantiomeric separation and sensitive determination of D,L-amino acids derivatized with fluorogenic benzofurazan reagents on Pirkle type stationary phases.
Enantiomeric separation and sensitive determination of D,L-amino acids derivatized with fluorogenic benzofurazan reagents on Pirkle type stationary phases.
复制标题
在 Pirkle 型固定相上用荧光苯并呋喃试剂衍生的 D,L-氨基酸的对映体分离和灵敏测定。
DOI：
10.1002/bmc.1130090103
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
K. Imai
中科院分区：
文献类型：
--
作者：
T. Fukushima;M. Kato;T. Santa;K. Imai
本文研究了4-氟-7-硝基-2，1，3-苯并恶二唑（NBD-F）、4-氟-7-硝基-2，1，3-苯并恶二唑（NBD-F）和4-氟-7-硝基-2，1，3-苯并恶二唑（NBD-F）衍生化D，L-氨基酸对映体的分离。N，N-二甲氨基磺酰基-7-氟-2，1，3-苯并恶二唑（DBD-F）和4-氨基磺酰基-7-氟-2，1，3-苯并恶二唑（ABD-F）在不同Pirkle型手性固定相上的高效液相色谱分析（CSP，Sumichiral OA系列），其中柠檬酸在甲醇中作为移动的相。由于最少的保留和没有分离观察到的外消旋苯丙氨酸甲酯，酰胺和药物的衍生物没有一个α-羧基，氨基酸衍生物的羧酸基团似乎有助于通过氢键上的N-酰基-氨基酸酰胺部分的CSP的衍生物的对映选择性固定。通过缬氨酸、苯甘氨酸、萘甘氨酸、萘乙胺或叔亮氨酸在CSP中的（S）或（R）构型实现衍生物的对映选择性保留。衍生物中的2，1，3-苯并恶二唑（苯并呋咱）部分有助于通过与芳香族部分（例如Pirkle型手性固定相中的3，5-二硝基苯基或萘基）的π-π相互作用来有效固定衍生物。生物样品（血清或脑匀浆）经甲醇脱蛋白和离心后，利用NBD-F衍生、对映体分离和荧光检测（530 nm em/470 nm ex），可方便地测定生物样品中的D-氨基酸。该方法的应用清楚地证明了以下结果：在健康志愿者的血清中检测到0.48-3.10 μ M水平的D-Ala。在骨髓瘤和需要肾透析的患者的血清中发现了D-Lys，在大鼠和牛的大脑中发现了D-Ser。通过使用不同类型的固定相进行峰鉴定，特别是那些具有相反构型的手性中心。
The enantiomeric separations of D,L-amino acids derivatized with fluorogenic reagents, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) and 4-aminosulphonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F) by high-performance liquid chromatography (HPLC) on various Pirkle type chiral stationary phases (CSPs, Sumichiral OA series) with citric acid in methanol as a mobile phase were studied. Since the least retention and no separation was observed for the derivatives of racemic phenylalanine methyl-ester, -amide and a drug without an alpha-carboxyl group, the carboxylic acid group of the amino acid derivatives seemed to contribute to the enantioselective fixation of the derivatives through hydrogen bonding on the N-acyl-amino acid amide moiety of the CSP. The enantioselective retention of the derivatives was attained through the (S) or (R) configuration of valine, phenylglycine, naphthylglycine, naphthylethylamine or the tert-leucine moiety in the CSP. The 2,1,3-benzoxadiazole (benzofurazan) moiety in the derivatives helps the effective fixation of the derivatives through a pi-pi interaction with an aromatic moiety such as a 3,5-dinitrophenyl or naphthyl group in the Pirkle type chiral stationary phases. D-Amino acids in biological samples were easily determined utilizing the present derivatization with NBD-F, enantiomeric separation and fluorometric detection (530 nm em/470 nm ex) following deproteinization of biological samples (serum or brain homogenate) with methanol and centrifugation. The applications of the method were clearly demonstrated by the following results; D-Ala was detected in sera of healthy volunteers at a level of 0.48-3.10 microM. D-Lys was found in the serum of a patient with myeloma and requiring renal dialysis, and D-Ser was found in rat and bovine cerebrum. Peak identification was performed by use of different types of stationary phases especially those bearing the opposite configuration to that of the chiral centre.