Yang Sen, Wu Li, He Yujian, Li Xiangjun
Department of Chemistry, University of Chinese Academy of Sciences, Beijing, 101408, China.
Department of Chemistry, University of Chinese Academy of Sciences, Beijing, 101408, China.
Anal Chim Acta. 2024 Sep 22;1323:343025. doi: 10.1016/j.aca.2024.343025. Epub 2024 Jul 30.
3-Hydroxyaspartate(3-HA) is a non-proteinaceous amino acid whose four stereoisomers have different biological functions, making it meaningful to separate and analyze them. A combination of capillary zone electrophoresis-mass spectrometry (CZE-MS) and electron circular dichroism enables the separation and analysis of the four stereoisomers of 3-HA, but relies on enantiomeric standards. In contrast, the new method based on vibrational circular dichroism (VCD) can successfully confirm the peak order of the four enantiomers simultaneously without a single enantiomeric standard. This method is suitable for molecules without UV absorption, as well as for molecules with polychiral centers, and does not require enantiomeric standards.
We present a new analytical method based on CZE-MS coupled with VCD for the simultaneous determination of four stereoisomers of 3-HA with two chiral centers (D-threo-3-hydroxyaspartate, D-THA; L-threo-3-hydroxyaspartate, L-THA; D-erythro-3-hydroxyaspartate, D-EHA; L-erythro-3-hydroxyaspartate, L-EHA) in the absence of an optically pure single enantiomeric standard. The semipreparations of the non-racemic mixtures of DL-THA and DL-EHA were performed using high performance liquid chromatography (HPLC), with enantiomeric excess values reaching 90 %. By comparing the experimental VCD spectra of the DL-THA/DL-EHA mixture with the theoretical VCD spectra of the single L-THA/L-EHA enantiomer, the configuration of the dominant enantiomer in the nonracemic mixture was determined, where the two characteristic peaks in the 1250-1750 cm spectral range fitted well. Finally, combined with the comparison of CE peak areas, the four stereoisomers were identified successfully.
This is the first combined CZE-MS and VCD method for the simultaneous separation and analysis of four stereoisomers of 3-HA without relying on enantiomeric standards. This method is simple and reliable, and provides a new idea for the separation and analysis of chiral compounds with polychiral centers, which are difficult to obtain from enantiomeric standards.
3-羟基天冬氨酸（3-HA）是一种非蛋白质氨基酸，其四种立体异构体具有不同的生物学功能，因此对其进行分离和分析具有重要意义。毛细管区带电泳-质谱联用（CZE-MS）和电子圆二色性相结合能够对3-HA的四种立体异构体进行分离和分析，但依赖于对映体标准品。相比之下，基于振动圆二色性（VCD）的新方法无需单一的对映体标准品即可成功同时确定四种对映体的峰顺序。该方法适用于无紫外吸收的分子以及具有多个手性中心的分子，且不需要对映体标准品。
我们提出了一种基于CZE-MS与VCD联用的新分析方法，用于在没有光学纯单一异构体标准品的情况下同时测定具有两个手性中心的3-HA的四种立体异构体（D-苏式-3-羟基天冬氨酸，D-THA；L-苏式-3-羟基天冬氨酸，L-THA；D-赤式-3-羟基天冬氨酸，D-EHA；L-赤式-3-羟基天冬氨酸，L-EHA）。使用高效液相色谱（HPLC）对DL-THA和DL-EHA的非外消旋混合物进行半制备，对映体过量值达到90%。通过将DL-THA/DL-EHA混合物的实验VCD光谱与单一L-THA/L-EHA对映体的理论VCD光谱进行比较，确定了非外消旋混合物中主要对映体的构型，其中在1250 - 1750 cm光谱范围内的两个特征峰拟合良好。最后，结合CE峰面积的比较，成功鉴定出四种立体异构体。
这是首次不依赖对映体标准品同时分离和分析3-HA的四种立体异构体的CZE-MS和VCD联用方法。该方法简单可靠，为难以从对映体标准品获得的具有多个手性中心的手性化合物的分离和分析提供了新思路。
