荧光标记DBD-脯氨酸的合成以及反相色谱分离手性胺的拆分效率。,Biomedical Chromatography

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荧光标记DBD-脯氨酸的合成以及反相色谱分离手性胺的拆分效率。
Biomedical Chromatography ( IF 1.8 ) Pub Date : 2004-10-08 , DOI: 10.1002/bmc.414
Jun Zhe Min ₁ , Toshimasa Toyo'oka , Masaru Kato , Takeshi Fukushima

Affiliation

由4-（N，N-二甲基氨基磺酰基）-7-氟-2,1,3-苯并恶二唑（DBD-F）反应合成了新型的荧光手性衍生试剂DBD-d（和l）-β-脯氨酸）和脯氨酸。合成的外消旋混合物通过手性固定相（CSP）色谱柱Chiralpak AD-H分离，流动相为正己烷-EtOH-TFA-二乙胺（70：30：0.1：0.1）。根据CSP色谱柱分离后从圆二色性（CD）检测器获得的结果确定dl形式。分馏的对映异构体与手性胺反应生成一对非对映异构体。在存在1-乙基-3-（3-二甲基氨基丙基）碳二亚胺（EDC）和吡啶作为活化剂的情况下进行标记。反应条件温和并且在非对映异构体形成过程中没有外消旋作用发生。所得非对映异构体在约570nm处发荧光（激发在约460nm处）。校正曲线在1-75 pmol范围内具有良好的线性，色谱图中的检出限小于1 pmol。将非对映异构体的可分离性与衍生自DBD-d（或l）-脯氨酸的非对映异构体进行了比较。从三种手性胺与DBD-d（或l）-β-脯氨酸的非对映异构体获得的拆分值（Rs）高于从DBD-d（或l）-脯氨酸获得的拆分值，例如dl-苯丙氨酸甲酯（dl- PAME），2.23比1.37；（R）（S）-1-苯基乙胺[（R）（S）-PEA]，2.09对1.13；（R）（S）-1-（1-萘基）乙胺[（R）（S）-NEA]，5.19对1.23。

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Synthesis of fluorescent label, DBD-beta-proline, and the resolution efficiency for chiral amines by reversed-phase chromatography.

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.

更新日期：2019-11-01

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