Li J, Waldron K C
Institute for Biological Sciences, NRC, Ottawa, ON, Canada.
Electrophoresis. 1999 Jan;20(1):171-9. doi: 10.1002/(SICI)1522-2683(19990101)20:1<171::AID-ELPS171>3.0.CO;2-6.
The separation of stereoisomers, particularly enantiomers, is important when their physiological activity differs. We have resolved the four stereoisomers each of alanylphenylalanine (Ala-Phe) and of leucylphenylalanine (Leu-Phe) by capillary electrophoresis using beta-cyclodextrin as a buffer additive and urea to enhance its solubility. A study of the influence of pH and beta-cyclodextrin concentration on the separations showed that weak inclusion complexes were formed between the dipeptides and chiral selector. It was found that pH could alter the migration order of enantiomers L-Ala-L-Phe and D-Ala-D-Phe, as well as L-Leu-L-Phe and D-Leu-D-Phe; however, there was no change in order for the other pairs of optical isomers. Electrophoretic mobility data were used to estimate the acid dissociation constants of the dipeptide isomers at pH < 7 with no chiral selector present. By varying the concentration of beta-cyclodextrin, the chiral selector, the binding constants of Ala-Phe and Leu-Phe optical isomers in their fully protonated and zwitterionic forms were estimated. For the four Ala-Phe stereoisomers, K = 42-66 M(-1) and 4-41 M(-1) for the cationic and zwitterionic forms, respectively. For the four Leu-Phe stereoisomers, K = 43-94 M(-1) and 1-28 M(-1) for the cationic and zwitterionic forms, respectively.
当立体异构体，特别是对映体的生理活性不同时，它们的分离就很重要。我们使用β-环糊精作为缓冲添加剂，并添加尿素以提高其溶解度，通过毛细管电泳分离了丙氨酰苯丙氨酸（Ala-Phe）和亮氨酰苯丙氨酸（Leu-Phe）各自的四种立体异构体。对pH值和β-环糊精浓度对分离影响的研究表明，二肽与手性选择剂之间形成了弱包合物。结果发现，pH值可以改变对映体L-Ala-L-Phe和D-Ala-D-Phe以及L-Leu-L-Phe和D-Leu-D-Phe的迁移顺序；然而，其他光学异构体对的顺序没有变化。在不存在手性选择剂的情况下，利用电泳迁移率数据估计了pH < 7时二肽异构体的酸解离常数。通过改变手性选择剂β-环糊精的浓度，估计了Ala-Phe和Leu-Phe光学异构体在其完全质子化和两性离子形式下的结合常数。对于四种Ala-Phe立体异构体，阳离子形式和两性离子形式的K分别为42 - 66 M⁻¹和4 - 41 M⁻¹。对于四种Leu-Phe立体异构体，阳离子形式和两性离子形式的K分别为43 - 94 M⁻¹和1 - 28 M⁻¹。
