Diastereoselective discrimination of lysine–alanine–alanine peptides by zwitterionic cinchona alkaloid-based chiral selectors using electrospray ionization mass spectrometry

Electrospray ionization-mass spectrometry (ESI-MS) was used to investigate stereoselective interactions between seven zwitterionic alkylsulfonate-modified cinchona alkaloid chiral selectors and biologically relevant lysine–alanine–alanine tripeptide and alanine–alanine dipeptide selectands in modified methanolic solutions. Ion intensities from full scan mass spectra were used to assess degrees of association, the ratios of which were used to calculate selectivities for different selector–selectand pairs. The results support prior work on similar systems using HPLC, in that binding is mediated in these systems primarily through the quinuclidine amine on the selector and the C-terminal carboxylate of the peptide. Nα- and Nα, Nɛ-acetylated forms of the tripeptide were used to study the relative contribution to binding imparted by the presence of multiple basic amines on the tripeptide with the selectors; this was not previously investigated by HPLC. The ability of the sulfonate group on the selector to reach and preferentially interact with the Nɛ-amine on the side chain of lysine was revealed. Overall, in acidic methanol conditions (0.5% acetic acid), degrees of association ranged from 1.5% to 17%, and selectivities ranged from non-selective to a 5.5:1 preference for binding one peptide stereoisomer over another with a given chiral selector. In sodium acetate (100 μM)-modified methanol solutions, significant changes in degrees of association (ranging from 4% to 25%) and selectivities (ranging from non-selective to 4.2:1 preference) were observed. These mass spectrometry experiments help to clarify the chiral recognition mechanism for these selectors and suggest that retention and selectivity could be further modulated in HPLC experiments through the utilization of alkali salt-containing mobile phases.

► Recognition mechanism of zwitterionic chiral selectors elaborated for peptides. ► Electrospray MS data complements prior HPLC work and adds new insight. ► Significant diastereoselective binding and discrimination for biorelevant peptides. ► Systematic variation of binding increments to elucidate binding mechanisms.