Péter Antal, Török Roland, Wright Karen, Wakselman Michel, Mazaleyrat Jean Paul
Department of Inorganic and Analytical Chemistry, University of Szeged, Dóm tér 7, H-6720 Szeged, Hungary.
J Chromatogr A. 2003 Dec 22;1021(1-2):1-10. doi: 10.1016/j.chroma.2003.09.015.
Direct and indirect high-performance liquid chromatographic (HPLC) methods were developed for the enantioseparation of spin-labelled, cyclic, chiral beta-amino acids containing nitroxide free radicals, trans-3-amino- 1-oxyl-2,2,5,5-tetramethylpyrrolidine-4-carboxylic acid (trans-POAC), cis-4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-3-carboxylic acid (cis-beta-TOAC) and their N-Fmoc-protected analogues, synthesized in racemic and enantiomerically pure forms. The direct method involved the use of a Chiralcel OD-RH column, while indirect separation was carried out by application of either 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate or (S)-N-(4-nitrophenoxycarbonyl)-phenylalanine methoxyethyl ester as chiral derivatizing agent. Use of 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey's reagent) as chiral derivatizing agent failed because of the low of yield of the derivatization reaction. Selection and variation of the mobile phase was restricted by the sensitivity of the spin-labelled amino acids to acidic conditions. Conditions affording the best resolution were found and the differences in separation capability of the methods were noted. The sequence of elution of the enantiomers was determined by different methods and, in the case of the beta-TOAC analogues, the absolute configurations of the enantiomers corresponding to each peak were identified.
开发了直接和间接高效液相色谱（HPLC）方法，用于对含有氮氧自由基的自旋标记环状手性β-氨基酸、反式-3-氨基-1-氧代-2,2,5,5-四甲基吡咯烷-4-羧酸（反式-POAC）、顺式-4-氨基-1-氧代-2,2,6,6-四甲基哌啶-3-羧酸（顺式-β-TOAC）及其N-Fmoc保护类似物进行对映体分离，这些化合物以外消旋和对映体纯形式合成。直接方法使用Chiralcel OD-RH柱，而间接分离则通过应用2,3,4,6-四-O-乙酰基-β-D-吡喃葡萄糖基异硫氰酸酯或（S）-N-（4-硝基苯氧基羰基）-苯丙氨酸甲氧基乙酯作为手性衍生化试剂来进行。使用1-氟-2,4-二硝基苯基-5-L-丙氨酸酰胺（Marfey试剂）作为手性衍生化试剂失败，因为衍生化反应产率低。自旋标记氨基酸对酸性条件的敏感性限制了流动相的选择和变化。找到了提供最佳分离度的条件，并注意到了这些方法在分离能力上的差异。通过不同方法确定了对映体的洗脱顺序，并且对于β-TOAC类似物，鉴定了与每个峰对应的对映体的绝对构型。
