High-Performance Liquid Chromatographic Separation of Stereoisomers of -Methyl-Substituted Unusual Amino Acids Utilizing Ion Exchangers Based on Alkaloids.

Novel peptides based on common amino acid building blocks may serve as possible drug candidates; however, their flexible structures may require stabilization via the incorporation of conformational constraints. The insertion of unusual amino acids is a feasible option that may provide improved pharmacokinetic and pharmacodynamic properties of such peptide-type drugs. The stereochemical purity of these kinds of building blocks must be verified by an efficient separation technique, such as high-performance liquid chromatography. Here, we present and discuss the results of the stereoselective separation mechanism of -methylated phenylalanine (-MePhe), tyrosine (-MeTyr), 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (-MeTic), and cyclohexylalanine (-MeCha) together with non-methylated Phe, Tyr, Tic, and Cha applying alkaloid-based chiral stationary phases (CSPs). The studied zwitterionic CSPs acting as ion exchangers provided optimal performance in the polar ionic mode when methanol or a mixture of methanol and acetonitrile was utilized as the mobile phase together with organic acid and base additives. It was found that the basicity of small amines applied as mobile phase additives did not directly influence the chromatographic ion exchange concept. However, the size of the amines and their concentration led to a reduced retention time following the principles of ion exchange chromatography. On the basis of a systematic study of the effects of the eluent composition on the chromatographic behavior, important structure-retention and enantioselectivity relationships could be revealed. Through a temperature study, it has become evident that the composition of the eluent and the structure of analytes markedly affect the thermodynamic properties.