Peptide Stereoisomer Separations

In a series of papers, the methodologies applied for amino acids were extended to peptide stereoisomer (enantiomer and diastereomer) separations [59,116,121-123], 

In general, stereoisomers do exist wherein n is the number of amino 

FIGURE 1.30 Micro-HPLC separation of all 4 stereoisomers of the dipeptide alanyl-alanine as FMOC derivatives (a) and DNP-derivatives (b), respectively, on a 0-9-(tert-butylcarbamoyl)quinine-based CSP. Experimental conditions Column dimension, 150 X 0.5 mm ID mobile phase (a) acetonitrile-methanol (80 20 v/v) containing 400 mM acetic acid and 4 mM triethylamine, and (b) methanol-0.5 M ammonium acetate buffer (80 20 v/v) (pHa 6.0) flow rate, 10 ixLmin temperature, 25 C injection volume, 250 nL detection, UV at 250 nm. (Reproduced fromC. Czerwenka et al., J. Pharm. Biomed. Anal., 30 1789 (2003). With permission.) 

In a follow-up study, the effect of introduction of achiral Gly residues into the Ala-peptide chain (di- and tripeptides) on the afforded enantiomer separations was thoroughly investigated [123]. One of the major findings was that the introduction of a Gly residue compromised the enantioselectivity in particular if the Gly residue was located at the /V-terminus. For example, a-values (for all-5/all-7 enantiomers) decreased in the order Ala-Ala Ala-Gly Gly-Ala for the DNB-protected dipeptide 

The enantiomer separation capability of the cinchonan carbamate selectors and CSPs, respectively, is extremely broad what acidic compounds is concerned (success rate close to 100% if the acidic functional group is close to the chiral center). Hence 

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Scriba, G. K. E. (2006). Recent advances in peptide and peptidomimetic stereoisomer separation by capillary electromigration techniques. Electrophoresis 11, 222-230. 

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. 

Separation of peptide stereoisomers can be accomplished using the same approach as for chiral amino acids, as mentioned before. 

Czerwenlra C, Lammerhofer M, Lindner W (2003) Micro-HPLC and standard-size HPLC for the separation of peptide stereoisomers employing an ion-exchange principle. J Pharm Biomed Anal 30 1789-1800... 

One of the problems attaching to the HPLC separation of peptides is the analysis of stereoisomers (enantiomers and diastereoisomers), that is, of peptides that differ only in the configuration of their amino acid residues. 

Numerous HPLC analyses have been carried out on biologically active diastereoisomeric peptides (encephalins, endorphins, hormones) in order to isolate them so that their activity can be studied however, the separation of stereoisomers is not yet common in the field of food science. 

J. Florance, High-performance liquid chromatographic separation of peptides and amino acid stereoisomers, J. Chromatogr., 414313 (1987). 

Florance J., Galdes A., Konteatis Z., Kosarych Z., Langer K., Martucci C., High Performance Liquid Chromatographic Separation of Peptide and Amino Acid Stereoisomers, Journal of Chromatography. 414 (1987), 313-323. 

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