The chiral separation of the LL- and DD-enantiomers of the dipeptides Ala-Tyr, Phe-Phe, and Asp-PheOMe has been investigated at pH 2.5 and pH 3.5 using beta-cyclodextrin (beta-CD), heptakis-(2,6-di-O-methyl)-beta-cyclodextrin, and heptakis-(2,3,6-tri-O-methyl)-beta-cyclodextrin as chiral selectors. According to electrospray mass spectrometry, heptakis-(2,6-di-O-methyl)-beta-cyclodextrin was a mixture of six isomers. Reversal of the enantiomer migration order upon increasing the buffer pH from 2.5 to 3.5 was observed for all peptides with beta-cyclodextrin, for Ala-Tyr and Phe-Phe in the presence of heptakis-(2,3,6-tri-O-methyl)-beta-cyclodextrin, and for Ala-Tyr using heptakis-(2,6-di-O-methyl)-beta-cyclodextrin. The migration behavior could be explained on the basis of the complexation constants and the mobilities of the peptide-cyclodextrin complexes. Both, the binding constants and complex mobilities decreased with increasing pH as the overall-charge of the peptides decreased. While the complexation constants primarily determined the migration order at pH 2.5, complex mobility dominated in most cases at pH 3.5.

The separation of dipeptide and tripeptide enantiomers using negatively charged single isomers as well as randomly sulfated and sulfonated cyclodextrins (CDs) was investigated with respect to the amino acid sequence of the peptides and the nature of the CDs. Standardized conditions concerning buffer pH and molarity, CD concentration, and separation voltage were applied. Compared to suffobutylether-beta-CD and heptakis-(2,3-dimethyl-6-sulfato)-beta-CD, randomly sulfated beta-CD as well as the single isomer derivatives heptakis-6-sulfato-beta-CD and heptakis-(2,3-diacetyl-6-sulfato)-beta-CD were the more universal CDs for enantioseparations. The enantiomer migration order depended to a greater extent on the CD than on the amino acid sequence of the peptide although small structural differences such as formation of a peptide amide or ester affected the chiral recognition by the randomly substituted CD derivatives. Using sulfobutylether-beta-CD or heptakis-(2,3-diacetyl-6-sulfato)-beta-CD the DD enantiomers migrated before the LL enantiomers for most peptides while the opposite migration order, i.e. LL before DD, was observed when heptakis-6-sulfato-beta-CD was applied as chiral selector.

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The present study was conducted in order to evaluate the cyclodextrin (CD)-mediated chiral separation of peptide enantiomers as uncharged analytes at pH 5.3 using randomly sulfated beta-cyclodextrin, heptakis-6-sulfato-beta-CD and heptakis-(2,3-diacetyl-6-sulfato)-beta-CD as chiral selectors. Although less effective compared to stronger acidic conditions, the CDs proved to be suitable chiral selectors for the present set of peptides at pH 5.3. The carrier ability of the negatively charged CDs upon reversal of the applied voltage may also be exploited leading to a reversal of the migration order. In addition, reversal of the enantiomer migration order upon increasing the buffer pH from 2.5 to 5.3 was also observed for Ala-Tyr in the presence of randomly sulfated beta-CD, for Ala-Phe, Ala-Tyr, Phe-Phe, Asp-PheNH(2) and Gly-Ala-Phe in the presence of heptakis-6-sulfato-beta-CD, and for Phe-Phe and Ala-Leu in the presence of heptakis-(2,3-diacetyl-6-sulfato)-beta-CD. The migration behavior could be explained on the basis of the complexation constants and the mobilities of the peptide-CD complexes. While a change in the affinity pattern of the CDs upon increasing the pH was observed for some peptides, complex mobility was the primary factor for other peptide-CD combinations affecting the enantiomer migration order at the two pH values studied.