Identification of 3-hydroxyaspartate with two chiral centers by capillary electrophoresis-mass spectrometry and vibrational circular dichroism independent of single enantiomer standard

Abstract

Background3-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. ResultsWe 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−1 spectral range fitted well. Finally, combined with the comparison of CE peak areas, the four stereoisomers were identified successfully. SignificanceThis 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.