Enantioseparation of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate tagged amino acids and other zwitterionic compounds on cinchona-based chiral stationary phases

Abstract

The fluorescent tag 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ Fluor reagent kit from Waters) is a commercial N-terminal label for proteinogenic amino acids (AAs), designed for reversed-phase separation and quantification of the AA racemates. The applicability of AQC-tagged AAs and AA-type zwitterionic compounds was tested for enantiomer separation on the tert-butyl carbamate modified quinine and quinidine based chiral stationary phases, QN-AX and QD-AX employing polar-organic elution conditions. The investigated test analytes included the enantiomers of the positional isomers of isoleucine (Ile), threonine, homoserine, and 4-hydroxyproline. Furthermore, β-AAs, cyclic, and heterocyclic AAs including trans-2-amino-cyclohexane carboxylic acid and trans-2-aminocyclohexyl sulfonic acid, phenylalanine derivatives substituted with halides with increasing electronegativity and 3,4-dihydroxyphenylalanine, cysteine-related derivatives including homocysteic acid, methionine sulfone, cysteine-S-acetic acid, and cysteine-S-acetamide as well as a small range of aminophosphonic acids were enantioseparated. A mechanistic interaction study of AQC-AAs in comparison with fluoresceine isothiocyanate-labeled AAs was performed. The chiral and chemoselective recognition processes involved in enantiomer separation and retention was systematically discussed. Special emphasis was set on the influential factors exhibited by the chemistry, branching position, and spatial properties of the investigated zwitterionic analytes. The general interest to separate and distinguish between different types of branched-chained AAs and metabolic side products thereof lies in the toxicity of some of these compounds, which makes for instance allo-Ile an attractive candidate in disease-related biomarker research.

Advanced dress-up chiral columns: New removable chiral stationary phases for enantioseparation of chiral carboxylic acids

“…Hellinger et al reported that interaction between AQC-AAs and QN-or QD-type CSPs can lead to great degrees of enantioseparation [25]. Because AQC-AA derivatives are more hydrophobic than N-Ac-AAs, we required larger MeOH contents in the mobile phase for their elution.…”

“…This tendency well agreed with previous study on enantioseparation of AQC-AAs with commercial cinchona alkaloid type CSPs (Chiralpak QN-AX and QD-AX). Hellinger et al discussed that poor enantioseparation of acidic AAs (Asp and Glu) derivatives with cinchona alkaloid CSPs lies in ion pairing interaction between the acidic side chain and the anion exchange moieties of CSPs [25]. The additional acidic group in b-and g-position of Asp and Glu functions as a competitor to the a-carboxylic acid for ion pairing interaction with the quinuclidine amine.…”

“…Several reports have appeared describing the chiral separations of AQC-derivatized AAs and peptides using cyclodextrin-bound CSPs [22][23][24]. Hellinger et al noted that noncovalent interactions between AQC-AAs and QNand QD-type CSPs can lead to high degrees of enantioseparation [25]. For highly sensitive and selective analyses of AQC-AAs, we also characterized them using liquid chromatography/tandem mass spectrometry (LC-MS/MS).…”

Novel chiral ionic liquids stationary phases for the enantiomer separation of chiral acid by high‐performance liquid chromatography

“…26 Among them, liquid chromatography is the most widely used. Liquid chromatography is based on the modified silica gel material comprising either immobilized small molecules of chiral selectors (CSs; eg, Cinchona-based ion exchangers), 27 macromolecule CSs (eg, biopolymers or synthetic polymers), 28 or macrocyclic CSs (cyclodextrin antibiotics). 29 The broad family of CSPs has enabled separation of almost any racemic mixture of choice, ranging from neutral lipophilic to highly polar hydrophilic compounds.…”

Design and synthesis of a novel pre-column derivatization reagent with a 6-methoxy-4-quinolone moiety for fluorescence and tandem mass spectrometric detection and its application to chiral amino acid analysis

“…As the chiral stationary phase, an enantioselective column QN-AX (1.5 mm i.d. × 150 mm, 25 • C, 5 m) was used, which has been reported to separate the enantiomers of N-protected amino acids efficiently[34].The 6-MOQ-Ala enantiomers were nicely separated within 15 min (˛ = 1.16) using 0.02% FA in MeCN-MeOH (50:50, v/v) solution as a mobile phase (200 L/min). The value of LLOD using fluorescence detection was 50 fmol/injection, and that of MS/MS was 0.1 fmol/injection.…”