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Discrimination of 4‑Hydroxyproline Diastereomers by Vibrational Spectroscopy of the Gaseous Protonated Species

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posted on 02.08.2012, 00:00 by Maria Elisa Crestoni, Barbara Chiavarino, Debora Scuderi, Annito Di Marzio, Simonetta Fornarini
Hydroxylation of proline is a prominent oxidative post-translational modification (oxPTM) in animals, characterized by site specificity and stereochemical control. The presence of this irreversible modification and the ensuing generation of a chiral center have been assayed in (2S,4R)-4-hydroxyproline and (2S,4S)-4-hydroxyproline forming the protonated species by electrospray ionization and sampling them by infrared multiple photon dissociation (IRMPD) spectroscopy. IRMPD spectra, recorded both in the 950–1950 cm–1 (using the CLIO free electron laser) and in the 3200–3700 cm–1 [using a tabletop parametric oscillator/amplifier (OPO/OPA) laser] regions, have been interpreted by comparison with the absorbance spectra of the lowest energy structures calculated at MP2/6-311+G** level of theory. Remarkable spectral differences have emerged in the fingerprint region, pointing to the unambiguous discrimination between S,R and S,S diastereomers. The main differences arise from the position of the carbonyl stretching mode, a signature of nonzwitterionic structures, moving from 1750 cm–1 for the S,R form to 1770 cm–1 for the S,S diastereomer. Furthermore, a well-defined band associated with the NH2 wagging mode at 1333 cm–1 is a distinct mark of the S,S isomer. Each gaseous protonated epimer comprises a population of at least three conformers, stabilized by intramolecular hydrogen bonds linking the two hydrogens of protonated secondary amine group with the 4-hydroxy substituent and with an oxygen atom of the carboxylic group, respectively. Interestingly, a tendency to adopt either C(4)-exo (up) or C(4)-endo (down) pyrrolidine puckering upon proline 4­(R)- or 4­(S)-hydroxylation, respectively, is observed here. The same bias is found in neutral hydroxyprolines and in collagen model peptides. In the protonated species under examination, this bias originates chirality-induced vibrational features revealed by IRMPD spectroscopy.

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