Derivatize, Racemize, and Analyze—an Easy and Simple Procedure for Chiral Amino Acid Standard Preparation for Enantioselective Metabolomics

Abstract

A simple, controllable, and reproducible stereoisomerization (racemization and epimerization) protocol for the preparation of scalemic α-amino acid mixtures from stereoisomerically pure standards was developed. Simply derivatize your amino acids with a racemization tag that incorporates a urea bond on the N-terminus of the target amino acid and incubate at elevated temperatures up to 95 °C for defined time periods until the targeted d-amino acid levels are obtained. The racemization tags investigated were 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC), aminophenyl-N-hydroxysuccinimidyl carbamate (AC), and 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APC). Employing this method, it was possible to create a ready-to-use, tailor-made chiral uniformly 13C and 15N labeled [U- 13C15N]-amino acid standard with the desired d-amino acid percentage within minutes or hours without sample cleanup. A racemization time of 30 min at 95 °C will lead to a d-amino acid level of 1–5%, while 6 h at 95 °C provides 15–30% d-amino acids. Racemization occurs due to imine formation at the chiral carbon atom bound to the urea-linking group without decomposition of labile amino acids such as Asn, Gln, Trp, Cit, and theanine. For amino acids possessing two chiral centers such as dl-Ile or dl-Thr, only the epimerization of isomers with different stereochemistry at the second chiral center will produce all four possible isobaric enantiomers. All measurements were performed on the zwitterionic Chiralpak ZWIX­(+) column using a dual hydro-organic flow gradient combined with HPLC-ESI-QTOF-MS analysis. This new racemization method solves the problem of (enantioselective) matrix effects and inaccurate results in LC-MS based enantioselective metabolomics and warrants full MS-compatibility.

Enantioselectivity Effects in Clinical Metabolomics and Lipidomics

“…To overcome this issue, a protocol to produce a tailored made internal standard with the desired d -amino acids level has been described together with a chromatographic procedure for AAs chiral separation with a zwitterionic Chiralpak ZWIX(+) column. These protocols should impact metabolomics analysis [ 26 ].…”

Recent Advances in Chiral Analysis of Proteins and Peptides

“…Indeed, Horak et al have compared the separation factor α for the enantioseparation of derivatized or underivatized common and uncommon DL-amino acids. This study has shown to the Chiralpak ZWIX (+) column offers a better separation to ACQ-DL-amino acids than underivatized amino acids, except for DL-Pro [92,93]. For this work, the authors do not study the DL-Cys.…”

“…The same group also compared both columns for the enantioseparation of ACQ-DL-amino acids. Conversely, the Chiralpak ZWIX (+) column allowed to obtain better separation factors α than the Chiralpak ZWIX (−), except for Pro, aThr, His and Arg [93]. Bajtai et al have also used Chiralpak ZWIX (+) to compare both zwitterionic columns for the enantioseparation of 20 dipeptide couples.…”

Discovery of Thanafactin A, a Linear, Proline-Containing Octalipopeptide from Pseudomonas sp. SH-C52, Motivated by Genome Mining

“…Preparation of AQC and amino acid derivatization with AQC were performed as described in the literature. 35 Enantioselective separation of amino acid-AQC derivatives were performed according to Horak et al 36 on a Chiralpak ZWIX (+) column using an Agilent 1290 UHPLC coupled with a SCIEX TripleTOF 5600+ ESI-MS instrument in positive ionization modes. Note that due to acidic hydrolysis with deuterated solvents, glutamine was deaminated to glutamic acid with HDX exchange to [Glu-[ 2 H] 2 + H] + : m/z 320.1210] (Figure S20).…”