Amino acids enantioseparation

Chapter 12 covers the important field of amino acid enantioseparation, in historical terms the first class of chiral compounds that were successfully separated by means of GC. The authors discuss direct enantioseparations with commercial and the noncommercial CSPs, the use of chiral modifiers of mobile phases, and derivatization of the amino acid antipodes followed by indirect separation. 

Early examples of enantioselective extractions are the resolution of a-aminoalco-hol salts, such as norephedrine, with lipophilic anions (hexafluorophosphate ion) [184-186] by partition between aqueous and lipophilic phases containing esters of tartaric acid [184-188]. Alkyl derivatives of proline and hydroxyproline with cupric ions showed chiral discrimination abilities for the resolution of neutral amino acid enantiomers in n-butanol/water systems [121, 178, 189-192]. On the other hand, chiral crown ethers are classical selectors utilized for enantioseparations, due to their interesting recognition abilities [171, 178]. However, the large number of steps often required for their synthesis [182] and, consequently, their cost as well as their limited loadability makes them not very suitable for preparative purposes. Examples of ligand-exchange [193] or anion-exchange selectors [183] able to discriminate amino acid derivatives have also been described. 

This report presents various methods developed primarily at our laboratory for chromatographic resolution of racemates of several pharmaceuticals (e.g., -blockers, NSAIDS, anta-acids, DL-amino acids, Bupropion, Baclofen, Etodolac, Carnitine, Mexiletine). Recently, we developed methods for establishing molecular dissymmetry and determining absolute configuration of diastereomers (and thus the enantiomers) of (/< .S )-Baclofcn, (/d.SJ-Bctaxolol with complimentary application of TLC, HPLC, H NMR, LCMS this ensured the success of diastereomeric synthesis and the reliability of enantioseparation. 

Enantioseparation Data for N-protected a-Amino Acids and Aryloxycarboxylic Acids on Butylcarbamates of Cs/Cg Stereoisomeric Cinchona Alkaloids... 

As previously observed, for most free amino acids and small peptides unbuffered hydroorganic mixtures are enough to yield good enantioseparations however, for some bifunctional amino acids and most other compounds, an aqueous buffer is usually necessary to enhance resolution. TEAA and ammonium nitrate are the most effective buffer systems, while sodium citrate was also effective for the separation of 2-arylpropionic acids (profens) on vancomycin CSPs [78], and ammonium acetate is the most widely used and appropriate in view of LC-MS applications (see Section 2.3.1.4). Small changes in ammonium acetate concentration of MeOH-water (90 10) mobile phases scarcely affected retention and— to a lesser extent—enantioselectivity of carnitine derivatives [61]. 

Direct and indirect chromatographic methods were developed and compared in systematic examinations for the enantioseparation of P-amino acids direct separation of underivatized analytes involved the use of commercially available Crownpak CR(-I-), teicoplanin, and ristocetin A CSPs [148], while indirect separation was based on precolumn derivatization with 2,3,4,6-tetra-G-acetyl-f)-D-glucopyranosyl isothiocyanate (GITC) or A - a-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (EDAA, Marfey s reagent), with subsequent separation on a nonenantioselective column. 

A comparative and comprehensive study on the enantioseparation of several unnatural P-amino acids has onlyjust been published in 2006, testing six commercially... 

Later, a commercially available TAG CSP was tested in the enantioseparation of 10 secondary a-amino acids, by using RP mobile mode systems [154]. The chromatographic results, compared with those obtained on a native teicoplanin CSP, were given as the retention, separation, and resolution factors, together with the enanti-oselective free energy difference corresponding to the separation of the investigated enantiomers. 

Petrusevska, K. et al.. Chromatographic enantioseparation of amino acids using a new chiral stationary phase based on a macrocyclic glycopeptide antibiotic, J. Sep. ScL, 29, 1447, 2006. 

Tesafova, E., Bosdkovd, Z., and Zuskova, L, Enantioseparation of selected iV-tert-butyloxycarbonyl amino acids in high-performance liquid chromatography and capillary electrophoresis with a teicoplanin chiral selector. J. Chromatogr. A, 879, 147, 2000. 

Grobuschek, N. et al., Enantioseparation of amino acids and drugs by CEC, pressure supported CEC, and micro-HPLC using a teicoplanin aglycone stationary phase, J. Sep. ScL, 25, 1297, 2002. 

Peter, A. et al.. High-performance liquid chromatographic enantioseparation of fS-amino acids, J. Chromatogr. A., 926, 229, 2001. 

Sztojkov-lvanov, A. et al., Comparison of separation efficiency of macrocyclic glycopeptide-based chiral stationary phases for the LC enantioseparation of fi-amino acids, Chromatographia, 64, 89, 2006. 

Berkecz, R. et al., LC enantioseparation of -lactam and P-amino acid stereoisomers and a comparison of macrocyclic glycopeptide and P-cyclodextrin-based columns, Chromatographia, 63, S37, 2006. 

Nimura N, Fujiwara T, Watanabe A, Sekine M, Furuchi T, et al. 2003. A novel chiral thiol reagent for automated precolumn derivatization and high-performance liquid chromatographic enantioseparation of amino acids and its application to the aspartate racemase assay. Anal Bio-chem 315 262-269. 

Enantioseparation of dansyl amino acids, free amino acids, hydroxy acids, and dipeptides... 

S Cladrowa-Runge, A Rizzi. Enantioseparations of 6-aminoquinolyl-lV-hydrox-ysuccinimidyl carbamate derivatized amino acids by capillary zone electrophoresis using native and substituted /3-cyclodextnns as chiral additives II. Evaluation of complexation constants. J Chromatogr A 759 167-175, 1997. 

In order to generally categorize the reaction schemes mentioned previously and the following ones in the course of indirect enantioseparation techniques, it has to be emphasized again, that the reciprocity principle should always be applicable. This means that if a chiral acid as the CDA can be used successfully to resolve the enantiomers of a chiral amine, then this optically pure amine as the CDA will equally well separate the enantiomers of the acid by the indirect method. The OPA reaction (see Figure 4) is therefore equally well suited for analyzing the optical purity of thiols, amines or amino acids. 

A Messina, AM Girelli, M Flieger, M Sinibaldi, P Sedmera, L Cvak. Direct resolution of optically active isomers on chiral packings containing ergoline skeletons. 5. Enantioseparation of amino acid derivatives. Anal Chem 68 1191-1196, 1996. 

Lammerhofer and Lindner [90] explained the chiral resolution of N-derivatized amino acids by CEC. The authors explained the formation of the transient diastereomeric ion-pairs between negatively charged analyte enantiomers and a positively charged chiral selector by multiple intermolecular interactions which might be differentially adsorbed to the ODS stationary phase. Furthermore, they claimed that the enantioseparation was achieved because of different observed mobilities of the analyte enantiomers originating from different ion-pair formation rates of the enantiomers and/or differential adsorption of the diastereoisomeric ion-pairs to the ODS stationary phase [90]. 

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