Direct High-Performance Liquid Chromatographic Separation of Peptide Enantiomers:  Study on Chiral Recognition by Systematic Evaluation of the Influence of Structural Features of the Chiral Selectors on Enantioselectivity

All-R/all-S enantiomers of oligoalanines (Ala_n, n = 1−10) with N-terminal protection group have been separated by HPLC on chiral stationary phases based on various cinchona alkaloid selectors. Structure−enantioselectivity relationships derived by extensive selector structure optimization provided insights into binding mechanisms and chiral recognition. Their interpretation was supported by X-ray crystal structures of amino acid and dipeptide, respectively, in complex with chiral selector. Optimized selectors have bulky elements representing steric barriers and deep binding pockets that afforded very high enantioselectivities; e.g., for the all-R and all-S enantiomers of N-(3,5-dinitrobenzoyl)alanylalanine, an α-value of 20.0 (corresponding to ΔΔG of −7.43 kJ/mol) was obtained with a chiral stationary phase based on 6‘-(neopentoxy)-9-O-tert-butylcarbamoylcinchonidine. Further, a chiral stationary phase based on 1,4-bis(9-O-quinidinyl)phthalazine was able to distinguish between the all-R and all-S enantiomers of hepta- to decaalanine peptides with enantioselectivity values between 1.8 and 1.9, corresponding to ΔΔG of −1.46 and −1.59 kJ/mol, respectively.