Chiral recognition of peptide enantiomers by cinchona alkaloid derived chiral selectors: mechanistic investigations by liquid chromatography, NMR spectroscopy, and molecular modeling.

The chiral recognition mechanism of a cinchona alkaloid based chiral selector for N-protected peptide enantiomers was investigated. A chiral stationary phase derived from this selector was employed for liquid chromatographic enantiomer separations. It showed exceptionally high enantiomer discrimination for the (all-R)- and (all-S)-enantiomers of dialanine (alpha = 20), while a pronounced loss of chiral recognition occurred upon the insertion of an additional alanine residue into the peptide backbone. This reduction of enantioselectivity was investigated in great detail by NMR spectroscopy of complexes of the chiral selector and the analyte enantiomers accompanied by molecular modeling studies. Investigation of intramolecular NOEs provided the conformational states of the free and complexed forms of the selector. The analysis of complexation-induced shifts yielded information on intermolecular interactions and allowed us to propose binding models, which were further supported by the observation of intermolecular NOEs, indicating the relative arrangements of selector and analytes. Stochastic molecular dynamics simulations were able to reproduce the chromatographic retention orders and energy differences, as well as the intermolecular NOEs. The computational data were used to evaluate the intermolecular forces responsible for analyte binding. In addition, the relative contributions of the fragments of the chiral selector to the enantioselective binding event were assessed. A spatial arrangement of the chiral selector and the analyte allowing the primary ionic interaction as well as hydrogen bonding and pi-pi-stacking to take place simultaneously was found to be essential to obtain very high enantioselectivities.