Norbert M. Maier

Separation of enantiomers: needs, challenges, perspectives.

Chiral drugs, agrochemicals, food additives and fragrances represent classes of compounds with high economic and scientific potential. First the present implications of their chiral nature and necessity of separating enantiomers are summarised in this article. In the following a brief overview of the actual approaches to perform enantioseparations at analytical and preparative scale is given. Challenging aspects of these strategies, such as problems associated with data management, choice of suitable chiral selectors for given enantioseparations and enhanced understanding of the underlying chiral recognition principles, are discussed. Alternatives capable of meeting the requirements of industrial processes, in terms of productivity, cost-effectiveness and environmental issues (e.g., enantioselective membranes) are critically reviewed. The impact of combinatorial methodologies on faster and more effective development and optimisation of novel chiral selectors is outlined. Finally, the merits and limitations of most recent trends in discrimination of enantiomers, including advances in the fields of sensors, microanalysis systems, chiroptical methods and chemical force microscopy are evaluated.

Towards ochratoxin A selective molecularly imprinted polymers for solid-phase extraction.

Molecularly imprinted polymers (MIPs) displaying selective binding properties for the mycotoxin ochratoxin A (OTA) in polar/protic media were prepared. Crucial to the success of these efforts was the implementation of rationally designed OTA mimics as templates and a set of novel basic and neutral functional monomers, allowing the maximization of the template-functional monomer association via ion-pairing, hydrophobic and steric interactions. MIPs prepared with a 20:1:1:3 molar ratio of cross-linking agent, template mimic, basic functional monomer and hydrophobic auxiliary monomer produced polymers with superior recognition properties compared to materials generated with other stoichiometries. Chromatographic evaluation using the OTA mimics, OTA and a set of structurally closely related compounds as analytes revealed pronounced substrate selectivity of these MIPs in polar/protic media, the templates and OTA being bound with significantly higher affinities. Complementary substrate selectivities/affinities were observed in aprotic and apolar solvents. The possibility of solvent-dependent tuning of substrate selectivity/affinity and the high binding capacity recommend the developed MIPs as promising solid-phase extraction adsorbents for clean-up and pre-concentration of OTA from various biologically relevant matrices.

Enantioselective anion exchangers based on cinchona alkaloid-derived carbamates: Influence of C8/C9 stereochemistry on chiral recognition

Four diastereomeric chiral stationary phases (CSPs) based on quinine, quinidine, epiquinine, and epiquinidine tert-butyl carbamate selectors were synthesized and evaluated under ion exchange HPLC conditions with a set of racemic N-acylated and N-oxycarbonylated alpha-amino acids as selectands. The enantioseparation potential of quinine- and quinidine-derived CSPs proved to be far superior to that of their C9-epimeric congeners. The absolute configuration of C9 stereogenic center of the cinchonan backbone of these selectors was identified as the structural feature controlling the elution order. Guided by an X-ray structure of a most favorable selector-selectand complex and the observed chromatographic enantioseparation data, a chiral recognition model was advanced. The contributions of ion-pairing, pi-pi donor-acceptor, hydrogen bonding and steric interactions were established as crucial factors.

Stationary phase-related investigations of quinine-based zwitterionic chiral stationary phases operated in anion-, cation-, and zwitterion-exchange modes.

The concept of recently introduced Cinchona alkaloid-type zwitterionic chiral stationary phases (CSPs) is based on fusing key cation- and anion-exchange (CX, AX) moieties in one single low-molecular mass chiral selector (SO) with the resulting CSPs allowing enantiomer separations of a wide range of chiral ionizable analytes comprising acids, bases, and zwitterionic compounds. Herein, we report principal, systematic investigations of the ion-exchange-type retention mechanisms available with the novel zwitterionic CSPs in nonaqueous polar organic mode. Typical CX and AX processes, corresponding to the parent single ion exchangers, are confirmed also for zwitterionic CSPs. Also the mechanism leading to recognition and retention of zwitterions was found to be ion exchange mediated in a zwitterion-exchange (ZX) mode. In both AX and CX modes the additional ionizable group within the SO besides the site responsible for the respective ion-exchange process could be characterized as an intramolecular counterion (IMCI) that effectively participates in the ion-exchange equilibria and thus, contributes to solute elution. In the ZX mode both oppositely charged groups of the zwitterionic SO were found not only to be the sites for simultaneous ion pairing with the analyte but also functioned as IMCIs at the same time. The main practical consequences of the IMCI feature were significant reduction of the amounts and even elimination of acidic and basic additives required in the eluent systems to afford analyte elution while still providing faster analysis than the parent single ion-exchanger-type CSPs. The set of ten structurally different zwitterionic CSPs employed in this study facilitated the establishment of correlations between chromatographic behavior of the CSPs with particular SO elements, thereby supporting the understanding of the working principles of these novel packing materials on a molecular level.

Direct liquid chromatographic enantioseparation of chiral α- and β-aminophosphonic acids employing quinine-derived chiral anion exchangers: determination of enantiomeric excess and verification of absolute configuration

Four types of quinine-derived chiral anion exchangers were used for the direct liquid chromatographic separation of enantiomers of chiral but N-protected α- and β-aminophosphonic acids. The respective and easily prepared 3,5-dinitrobenzyloxycarbonyl and dinitrophenyl derivatives were well separated, yielding α-values between 1.1 and 2.8 and resolution values in the range 2.0–9.0. Besides the chromatographic investigations, data on enantiomeric excesses (ees) and the absolute configurations of some enantiomerically enriched α- and β-aminophosphonic acids prepared by stereoselective synthesis were also determined. The results obtained by this method (ee and absolute configuration) correspond well with the results previously obtained by a 31P-NMR spectroscopic method.

Diphenylethanediamine derivatives as chiral selectors V1. Efficient normal-phase high-performance liquid chromatographic enantioseparation of underivatized chiral arylalcohols on four differently linked 3,5-dinitrobenzoyldiphenylethanediamine-derived chiral stationary phases☆

Abstract Four brush-type chiral stationary phases (CSPs) have been prepared, via immobilization of ( R , R )-3,5-dinitrobenzoyl-1,2-diphenylethane-1,2-diamine (DNB-DPEDA) with structurally different anchoring groups. These phases separate at normal-phase HPLC conditions the enantiomers of numerous underivatized chiral aromatic alcohols, including ArCH(OH)R, ArCH 2 CH(OH)R, Ar(CH) 2 CH(OH)R, simple tertiary arylalkylcarbinols and trans -2-arylcyclohexanols. In mobile phases of low polarity separations are characterized by considerate levels of enantioselectivity ( α = 1.1–2.7), excellent band shapes and short elution times. The hydroxyl group of the analytes is shown to be essential for stereodiscrimination, while π-basicity and steric bulkiness determine the magnitude of enantioselectivity. For simple structured arylalkylcarbinols a correlation is found between elution order and absolute configuration of the analyte. A rationale for molecular recognition of carbinol type analytes on DNB-DPEDA-derived CSPs is advanced. Their general applicability is demonstrated for a number of chiral auxiliaries, solvating agents and drug intermediates of carbinol structure.

Diphenylethanediamine derivatives as chiral selectors VIII. Influence of the second amido function on the high- performance liquid chromatographic enantioseparation characteristics of (N-3,5-dinitrobenzoyl)-diphenylethanediamine based chiral stationary phases

Abstract The effect of the second amido group in ( R , R )- and ( R , S )-3,5-dinitrobenzoylated 1,2-diphenylethane-1,2-diamine (DPEDA) derived chiral selectors was investigated. Structurally related mono-amidic (“deaza”) model compounds were synthesized and immobilized. The HPLC performance of the resultant two new chiral stationary phases (CSPs) was compared with the broadly applicable diamidic analogues. On the “deaza” CSPs special enantioseparation capabilities for underivatized alcohols and carboxylic acids were significantly reduced whereas separation of amides, ureas and carbamates and also analytes containing ester functions were substantially improved.

Unexpected enantioseparation of mandelic acids and their derivatives on 1,2,3-triazolo-linked quinine tert-butyl carbamate anion exchange-type chiral stationary phase.

Replacement of the flexible thioether linker for the novel, rigid 1,2,3-triazole spacer group in the course of immobilization of quinine tert-butyl carbamate onto a silica surface led to a chiral stationary phase (CSP) with enhanced enantioselectivity for the resolution of mandelic acid and derivatives thereof. These new CSPs allowed efficient resolution of a wide set of mandelic acids with α-values between 1.08 and 1.68. The high loadability of these chiral ion exchange type CSPs allows preparative separation in the milligram range on an analytical column of 100×4 mm id in a single run as it was demonstrated for 4-trifluoromethylmandelic, 2-naphthylglycolic and 3,4-methylenedioxymandelic acids. The chiral recognition process has been studied using a library of 25 diverse racemic probes. A tentative model suggests that the rigid 1,2,3-triazole group takes part in the formation of an enantioselective-binding pocket of the entire and immobilized selector moiety of the CSP. The primary interaction site is given by the ionizable quinuclidine group of the Cinchona alkaloid supported by possible π-π stabilization effects within the selector-selectand complex.

Enantioselective liquid-solid extraction for screening of structurally related chiral stationary phases

SummaryAn enantioselective liquid-solid batch extraction method is described for the screening of novel chiral stationary phases (CSPs) during optimization studies of chiral selectors derived form a common lead structure. Extraction enantioselectivity (α) values can be calculated from the enantiomeric excess ee-values of the selectand, which are measured in the liquid phase by enantioselective HPLC. Extraction α-values have been correlated with chromatographic α-values. The influence was studied of several experimental parameters of the assay (pHa, buffer concentration, temperature, selector/selectand and phase ratio) on the enantiomeric excess (ee) values of the selectands and the enantioselectivity of the CSPs, respectively. The derived statistically significant model has then been implemented to predict chromatographic α-values of novel CSPs. For example, an ee of 89.3% for DNB-Leu as selectand could be achieved in batch extraction for a novel synthesized but mechanistically similarly-acting CSP derived form quinine. This corresponds to a calculated extraction α-value of 17.7. Based on this αextraction a chromatographic α-value of 28.8 was predicted by the linear correlation model; the experimental HPLC α-value of 31.7 was in good agreement and demonstrated the validity of the proposed screening method. The method is particularly helpful in SO optimization studies.

Triazolo-linked cinchona alkaloid carbamate anion exchange-type chiral stationary phases: Synthesis by click chemistry and evaluation.

Immobilization strategy based on Huisgen 1,3-dipolar cycloaddition (click chemistry) of 10,11-didehydrocinchona tert-butylcarbamates to azido-grafted silica gels has been evaluated for preparation of novel chiral stationary phases (CSP 1-3). The resultant 1,2,3-triazole-linked CSPs were tested under various mobile phase conditions (polar organic and reversed phase mode) with a representative set of structurally diverse racemic acids including N-protected aminoacids, aromatic and aryloxycarboxylic acids as well as binaphthol phosphate. The chiral recognition performance of the C3-triazole-linked CSPs was found to mirror largely that of the known C3-thioether-linked CSP in terms of elution order, enantioselectivity and retention behavior. In an effort to assess the non-specific binding expressed as retention increment of these triazole-linked CSPs, the parent azidopropyl- and triazole-modified silica materials (thus not containing the chiral head ligand) were studied independently. Compared with the corresponding CSPs, the analyte retention on the azidopropyl control column was very low, and practically negligible on the corresponding triazole-modified reference column. Only minor losses in analyte retention behavior (<5%) were observed with triazole-linked CSPs after two month of continuous use with polar-organic and reversed-phase-type mobile phases, highlighting the excellent stability of the 1,2,3-triazole linker.