Amino acid enantiomers, chiral derivatization

H Brucker, M Langer, M Lupke, T Westhauser, H Godel. Liquid chromatographic determination of amino acid enantiomers by derivatization with o-phthalaldehyde and chiral thiols. Applications with reference to food science. J Chromatogr A 697 229-245, 1995. 

Conversion of amino acids to produce a diaster-eoisomeric peptide by reaction with an N-carboxy-anhydride, e.g., L-Phe-N-carboxy-anhydride, has been used for determination of optical purity using RPC. Alternatively, for analysis of amino acid enantiomers without derivatization, two options are available (1) chiral mobile phases such as the N,N-di- -propyl-L-alanine-copper(n) complex can be used with reversed-phase columns, and (2) stationary phases with a covalently bound ligand capable of stereo recognition can be used. Such ligands include cyclodextrins, albumins, glycoproteins, and copper(II) complexes. 

One of the most useful applications of chiral derivatization chromatography is the quantification of free amino acid enantiomers. Using this indirect method, it is possible to quantify very small amounts of enantiomeric amino acids in parallel and in highly complex natural matrices. While direct determination of free amino acids is in itself not trivial, direct methods often fail completely when the enantiomeric ratio of amino acid from protein hydrolysis must be monitored in complex matrices. 

S Einarsson, B Josefsson, P Moller, D Sanchez. Separation of amino acid enantiomers and chiral amines using precolumn derivatization with (+)-l-(9-fluorenyl)ethyl chlo-roformate and reversed phase liquid chromatography. Anal Chem 59, 1191, 1987. 

Amino acid enantiomers can be separated on a chiral stationary phase after derivatization with chloroformates (Abe et al., 1996). The derivatization procedure is quite simple and rapid, but the derivatizing reagent must be synthesized, which complicates the assay. Another method for the analysis of amino acid enantiomers uses N,0-pentafluoropropionyl isopropyl derivatives and a chiral column with NPD detection (Hashimoto et al., 1992). 

Abe I, Fujimoto N, Nishiyama T, Terada K, Nakahara T. 1996. Rapid analysis of amino acid enantiomers by chiral-phase capillary gas chromatography. J Chromatogr A 722 221. Ahuja S. 1976. Derivatization in gas chromatography. J Pharm Sci 65 163. 

Three approaches can be employed to separate peptide stereoisomers and amino acid enantiomers separations on chiral columns, separations on achiral stationary phases with mobile phases containing chiral selectors, and precolumn derivatization with chiral agents [111]. Cyclodextrins are most often used for the preparation of chiral columns and as chiral selectors in mobile phases. Macrocyclic antibiotics have also been used as chiral selectors [126]. Very recently, Ilsz et al. [127] reviewed HPLC separation of small peptides and amino acids on macrocyclic antibiotic-based chiral stationary phases. 

There are two major approaches to achieve enantiomeric separation of d- and L-amino acids. The first involves precolumn derivatization with a chiral reagent, followed by RP-HPLC [226], while the second involves direct separation of underivatized enantiomers on a chiral bonded phase [227], Weiss et al. [209] determined d- and L-form of amino acids by applying derivatization with OPA and chiral /V-isobutyryl-L-cysteine. 

N Nimura, T. Kinoshita. o-Phthalaldehyde-A -acetyl-t.-cystcine as a chiral derivatization reagent for liquid chromatographic optical resolution of amino acid enantiomers and its application to conventional amino acid analysis. J Chromatogr 352 169-177, 1986. 

Chirality of derivatized cyclodextrin was used for recognition of stereoisomers. Phenylazobenzoyl modified y-cyclodextrin was anchored onto silica gel used as stationary phase in HPLC and photoresponsive chromatographic behavior of dansyl amino acid enantiomers was studied [64],... 

Owing to the different biological activity of D- and L-enantiomers of seleno-amino acids, the chiral separation of optical isomers has been undertaken in sele-nized yeast and in yeast-based commercial supplements. Both, chiral stationary phase (crown ether) and chiral derivatization prior to reversed-phase HPLC were used [16, 77, 78],... 

Anal3des derivatized with a chiral derivatization reagent have been separated by reversed-phase liquid chromatography " the reversed-phase liquid chromatography of derivatized amino acid enantiomers was analyzed using... 

R. H. Buck and K. Krummen, Resolution of amino acid enantiomers by high-performance liquid chromatography using automated pre-column derivatization with a chiral reagent, /. Chromatogr., A, 1984, 315, 279-285. 

This is the indirect approach for the chromatographic separation of enantiomers. The reaction of the two forms of an enantiomer with an optically pure chiral reagent gives a mixture of diastereoisomers that are not mirror images of each other and therefore can be separated on a nonchiral, common LC phase. This is shown in Figure 4 where amino acids have been derivatized with o-phthaldialdehyde and N-isobutyryl-... 

The indirect method is an efficient technique for the enantioseparation of amino acids. However, it is essential that the chiral derivatization reaction proceeds completely in both enantiomers, and that the racemization reaction does not occur. Furthermore, if the optical purity of the derivatization reagent is not known, and/or is not taken into consideration, the optical purity of the amino acid will not be determined precisely. Thus, the indirect method is unsuitable for the analysis of amino acid enantiomers in a standard sample and pharmaceutical preparations, where a low amount of antipode, at a level of 0.1 or 0.05%, should be determined. 

Many chiral derivatization reagents have been developed for the enantioseparation of amino acids wherein ultraviolet-visible or fluorescence tags are introduced. The fluorescence derivatization is more effective for the determination of amino acid enantiomers in complex matrices in terms of sensitivity and/or selectivity. Table 1 shows the chiral derivatization reagents, whose structures are shown in Figure 2, used for the enantioseparation of amino acids. 

The need to resolve the d- and L-enantiomers of amino acids has grown in recent years as it has been recognized that they differ in biological and physicochemical properties. Amino acid enantiomers cannot be resolved in achiral systems it is therefore necessary to have a second chiral center either in the chromatographic system or to create one by derivatization in the molecule to be separated (Figure 2). 

Disz I, Berkeez R, Peter A. Application of chiral derivatizing agents in the high-performance liquid chromatographic separation of amino acid enantiomers a review. J Pharm Biomed Anal 2007 47 1—15. 

FIGURE 6.5 Reaction of chiral derivatization reagents with amino acid enantiomers. 

It is because the chiral selector was a relatively simple molecule having naturally its stereoisomer that the chiral recognition mechanism could be fully established in the case of DNB-derivatized amino acid enantiomer separation. Most chiral selectors are very complicated molecules making extremely difficult to predict a priori a chiral recognition mechanism. 

Bertrand M, Chabin A, Brack A, Westall F, Separation of amino acid enantiomers via chiral derivatization and non-chiral gas chromatography. J. Chromatogr. A 2008 1180 131-137. 

MOST POPULAR CHIRAL REAGENTS USED FOR DERIVATIZATION OF AMINO ACID ENANTIOMERS... 

The method was further extended for analysis of d- and L-amino acids in mouse kidney in a similar manner. HPLC methods, for the resolution of amino acid enantiomers that utilized a chiral stationary phase or a chiral mobile phase additive were considered expensive and less satisfactory due to the high retention results in broad peaks. The separation of FDAA derivatives followed by HPLC, as described above, was found to be much more successful as there was neither needed a post column derivatization nor fluorimetric analysis and moreover a subnanomolar sensitivity was attained [33], d- and L-enantiomers of glutamate, aspartate, asparagine, serine, threonine, alanine, proline, tyrosine, valine, methionine, isoleucine, leucine, phenylalanine, and histidine were derivatized with FDAA and the diastereomers were separated by 2D TLC. Each was separated except for the two spots comprising Tyr and Val, and He, Leu, and Phe. Only histidine was separated further into D- and L-diastereomers by TLC. The excess hydrolyzed FDAA moved to the front... 

A simple and rapid method of separating optical isomers of amino acids on a reversed-phase plate, without using impregnated plates or a chiral mobile phase, was described by Nagata et al. [27]. Amino acids were derivatized with /-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA or Marfey s reagent). Each FDAA amino acid can be separated from the others by two-dimensional elution. Separation of L- and D-serine was achieved with 30% of acetonitrile solvent. The enantiomers of threonine, proline, and alanine were separated with 35% of acetonitrile solvent and those of methionine, valine, phenylalanine, and leucine with 40% of acetonitrile solvent. The spots were scraped off the plate after the... 

---