Questioning chirality in therapeutic peptides: effect of Asp, His and Gly? – GLYMS

Due to different biological properties of enantiomers, the control of chirality of drug is an issue of utmost importance in pharmaceutical industry. For therapeutic peptides, partial epimerization of constitutive L-amino acid into their D form represents an acute matter of concern in the monitoring of synthetic impurities. Aspartic acid and histidine are prone to such optical conversion that can happen during synthesis and also after administration upon action of L-to-D isomerase. Checking the homochirality of synthetic peptides throughout the whole drug development process implies the efficient separation of diasteroisomeric sequences at very low level of contamination (<0.1%) which constitutes a very significant analytical challenge. Although much effort has been devoted up to now to chiral separations of enantiomers or diastereoisomers upon liquid chromatography (LC) and capillary electrophoresis (CE) on one hand, and mass spectrometry (MS) on the other hand, such approaches failed to provide the required sensitivity and often lack versatility. The objective of the GLYMS project is to develop an integrative approach combining comprehensive chiral LC/CE separation hyphenated to tandem mass spectrometry (MS/MS) enabling Ion Mobility (IM) and Energy-Resolved Mass Spectrometry experiments on protonated and cationized molecular ions to detect and quantify trace amount of residue epimerization in model tripeptides. The sequences of interest will be designed to contain aspartic acid (Asp) and histidine (His) in both optical forms at various positions. Glycine (Gly), the sole achiral amino acid, will also be investigated as reference to probe specific gas-phase dissociation behaviors due to the lack of side-chain to be compared to the fragmentation patterns of L- or D-Asp/His containing sequences displaying opposite pendant chain orientations. A home-made library of more than 300 peptides is available to validate the proof of concept on more complex sequences.