Min Jun Zhe, Toyo'oka Toshimasa, Kato Masaru, Fukushima Takeshi
Department of Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan.
Biomed Chromatogr. 2005 Jan;19(1):43-50. doi: 10.1002/bmc.414.
DBD-d(and l)-beta-proline, new fluorescent chiral derivatization reagents, were synthesized from the reaction of 4-(N,N-dimethylaminosulfonyl)-7- fl uoro-2,1,3-benzoxadiazole (DBD-F) with beta-proline. The racemic mixture synthesized was separated by a chiral stationary phase (CSP) column, Chiralpak AD-H, with n-hexane-EtOH-TFA-diethylamine (70:30:0.1:0.1) as the mobile phase. The dl-forms were decided according to the results obtained from a circular dichroism (CD) detector after separation by the CSP column. The fractionated enantiomers reacted with chiral amine to produce a couple of diastereomers. The labeling proceeded in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and pyridine as the activation reagents. The reaction conditions were mild and no racemization occurred during the diastereomer formation. The resulting diastereomers fluoresced at around 570 nm (excitation at around 460 nm). Good linearity of the calibration curves was obtained in the range 1-75 pmol and the detection limits on chromatogram were less than 1 pmol. The separability of the diastereomers was compared with the diastereomers derived from DBD-d(or l)-proline. The resolution values (Rs) obtained from the diastereomers of three chiral amines with DBD-d(or l)-beta-proline were higher than those derived from DBD-d(or l)-proline, e.g. dl-phenylalanine methylester (dl-PAME), 2.23 vs 1.37; (R)(S)-1-phenylethylamine [(R)(S)-PEA], 2.09 vs 1.13; and (R)(S)-1-(1-naphthyl)ethylamines [(R)(S)-NEA], 5.19 vs 1.23. The results suggest that the position of COOH group on pyrrolidine moiety in the structures is one of the important factors for the efficient separation of a couple of the diastereomers.
新型荧光手性衍生试剂DBD-d（和l）-β-脯氨酸由4-（N，N-二甲基氨基磺酰基）-7-氟-2,1,3-苯并恶二唑（DBD-F）与β-脯氨酸反应合成。合成的外消旋混合物通过手性固定相（CSP）柱Chiralpak AD-H进行分离，以正己烷-乙醇-三氟乙酸-二乙胺（70:30:0.1:0.1）作为流动相。根据通过CSP柱分离后从圆二色性（CD）检测器获得的结果确定dl-形式。分馏得到的对映体与手性胺反应生成一对非对映体。标记反应在1-乙基-3-（3-二甲基氨基丙基）碳二亚胺（EDC）和吡啶作为活化试剂的存在下进行。反应条件温和，在非对映体形成过程中没有发生消旋化。得到的非对映体在约570nm处发荧光（激发波长约为460nm）。在1-75pmol范围内校准曲线具有良好的线性，色谱图上的检测限小于1pmol。将非对映体的可分离性与由DBD-d（或l）-脯氨酸衍生的非对映体进行比较。由三种手性胺与DBD-d（或l）-β-脯氨酸得到的非对映体的分离度值（Rs）高于由DBD-d（或l）-脯氨酸衍生的非对映体，例如dl-苯丙氨酸甲酯（dl-PAME），分别为2.23和1.37；（R）（S）-1-苯乙胺[（R）（S）-PEA]，分别为2.09和1.13；以及（R）（S）-1-（1-萘基）乙胺[（R）（S）-NEA]，分别为5.19和1.23。结果表明，结构中吡咯烷部分上COOH基团的位置是有效分离一对非对映体的重要因素之一。
