Cyclodextrins are well known for their ability to separate enantiomers of drugs, natural products, and other chiral substances using HPLC, GC, or CE. The resolution of the enantiomers is due to the formation of diastereomeric complexes between the cyclodextrin and the pairs of enantiomers. The aim of this study was to determine the binding constants of the complexes between alpha- and beta-cyclodextrin and the enantiomers of a series of aliphatic and aromatic amino acids, and dipeptides, using a potentiometric titration method. The results of this method are compared to other methods, and correlated to findings in cyclodextrin-modified capillary electrophoresis and possible complex structures. Potentiometric titration was found to be an appropriate tool to determine the binding constants of cyclodextrin inclusion complexes.

Cyclodextrins (CDs) are often used to separate the enantiomers of chiral drugs by HPLC, GC, and capillary electrophoresis (CE). For dipeptides containing one aromatic amino acid, a reversal of the migration order is observed in CE upon increasing the pH value of the background electrolyte from 2.5 to 3.5. In order to understand the mechanism of chiral recognition of this phenomenon, NMR experiments, namely complexation‐induced chemical shifts (CICS) and ROESY, were performed. Comparing the CICSs obtained for the pairs Diac‐β‐CD/Ala‐Phe and HDAS‐β‐CD/Ala‐Phe and Diac‐β‐CD/Ala‐Tyr and HDAS‐β‐CD/Ala‐Tyr revealed very small changes, thereby indicating a rather weak interaction of the respective guest molecules with their host. The CICS for β‐CD and HS‐β‐CD confirmed an inclusion of the aromatic moiety into the CD cavity. It is shown that, at pH 2.5, the D,D‐enantiomer of Ala‐Tyr immerses deeper into the cavity of β‐cyclodextrin than the L,L‐enantiomer, and this was subsequently confirmed by molecular dynamics simulations. Furthermore, at pH 3.5 the immersion is shallower, as shown by the 1H and ROESY NMR findings and confirmed by the molecular dynamics simulations. The immersion depths of the dipeptides were calculated by defining a plane in the cavity to gain information about the different behaviour of the studied chemical species. The employed procedure is easily generalised to other host‐guest complexes and is expected to improve data evaluation in other cases, too. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

The separation of dipeptide and tripeptide enantiomers using a neutral single isomer cyclodextrin (CD) derivative, heptakis-(2,3-di-O-acetyl)-beta-CD (DIAC-beta-CD), was investigated with respect to the amino acid sequence applying standard separation conditions. With only one exception the DD-enantiomers migrated faster than the LL-stereoisomers. Separations obtained for the same set of peptides using beta-CD and the sulfated single isomer derivatives heptakis-(2,3-di-O-acetyl-6-sulfo)-beta-CD (HDAS-beta-CD) and heptakis-6-sulfo-beta-CD (HS-beta-CD) revealed identical enantiomer migration order in the presence of the 2,3-disubstituted derivatives DIAC-beta-CD and HDAS-beta-CD. In contrast, reversed migration sequence was found for beta-CD and HS-beta-CD compared to DIAC-beta CD and HDAS-beta-CD indicating the importance of the substitution pattern on the wider rim of the CD cavity on the chiral recognition of the peptide enantiomers by the CDs. Nuclear magnetic resonance (NMR) experiments indicated different complexation modes between the enantiomers and the CDs depending on the presence of acetyl substituents on the wider rim of the CD torus. Thus, the CD-induced chemical shifts observed in samples containing Ala-Phe or Ala-Tyr and beta-CD or HS-beta-CD were consistent with an inclusion of the aromatic moiety into the CD cavity. Although the CD-induced chemical shifts in the presence of DIAC-beta-CD and HDAS-beta-CD did not allow direct conclusions on the complexation mode they substantially differed from those observed in the presence of 2,3-unsubstituted CDs indicating different structures of the peptide-CD complexes.

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