Chromatography and mass spectrometry in the study of structure and dynamics of chiral molecules

Part A Dynamic high performance liquid chromatography on chiral stationary phases is a consolidated technique that allows the investigation of chiral molecules with labile stereogenic elements that interconvert very quickly at room temperature and result in stereoinversion processes occurring on the time scale of the separation process. Kinetic parameters for on-column interconversions can be extracted from exchange-deformed experimental peak profiles by computer simulation. The technique has been used in a wide range of temperatures and is complementary in scope to dynamic nuclear magnetic resonance spectroscopy. Here we report, in the first part, the separation of the enantiomers of benzodiazepines, a class of molecules whose conformational enantiomers interconvert not only through a single bond rotation but also via “ring-flip” inversion. The second part concerns the first HPLC resolution of the conformational enantiomers of tri-O-thymotide (TOT) a macrocyclic trilactone existing in fast-exchanging multiple chiral conformations. Variable chromatography on brush type stationary phases showed dynamic features due to on-column interconversions of TOT. Part B Chiral recognition is a branch of chemistry aimed at understanding the reactivity as well as the size- and shape-specificity of non-covalent interactions between molecular aggregates formed by chiral species. Mass spectrometry (MS) is a powerful tool for investigating chiral recognition in the gas phase in the absence of perturbing environmental phenomena and discriminating and even quantifying chiral species by interaction with chiral reference molecules. A small library of synthetic receptors was prepared by macrocyclization of complementary A and B fragments to yield A2B2 macrocycles, where A are activated forms of isophthalic acid derivatives and B are chiral, C2 symmetric 1,2-diamines derived from 1,2-diphenylethylendiamine. Their enantioselectivity was investigated by ESI-MS and revealed large enantioselectivities towards the enantiomers of aminoacids and peptide guests. The stability of the complexes increases with the size of guests and with large aromatic portions on the guest. Part C This part of my academic program was developed at the University of California, Davis, under the supervision of prof. Carlito Lebrilla, during the last period of my Ph.D. activity. An important goal in proteomic is to quantify the profile changes of protein abundances in biological systems. Quantifying these changes is a key to understand changes in cell state at a molecular level. In the last years label-free quantitation using MRM (multiple reaction monitoring), which correlates the mass spectrometric signal of intact proteolytic peptides with the relative or absolute protein quantity, is become a powerful tool for accurate quantitation. Here we report a label-free method to quantify proteins and their glycoforms in biologic fluids (milk, feces and urine) by MRM, including the selection of proteotryptic peptides and the optimization and validation of transitions.