Isolation and identification of peptide conformers by reversed-phase high-performance liquid chromatography and NMR at low temperature

Abstract

Peptide conformers with one or more rotationally hindered peptide bonds due to the presence of proline and/or another N-substituted amino acid residue in the molecule were separated by reversed-phase chromatography at low temperatures, isolated and identified by NMR. The scope of this investigation included the cis-trans isomers of the dipeptides Leu-Pro, Phe-Pro and Tyr-Pro as well as conformers of opioid peptides containing proline and/or the proline-like Tic (1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid) residues: Tyr-Pro-Phe (β-casomorphin 1–3 fragment), Tyr-Tic-Phe-Phe, Tyr-Pro-Phe-Pro-Gly (β-casomorphin-5), Tyr-Tic-Phe-Phe-Val-Val-Gly-NH 2 and Tyr-Tic-Phe-Gly-Tyr-Pro-Ser-NH 2. Chromatography with micropellicular and totally porous octadecylated silica stationary phases and aqueous methanol under isocratic elution conditions resulted in well separated peaks of the rotational isomers at sufficiently low temperatures. Preparative RP-HPLC was carried out with eluents containing water and methanol, both deuterated, and the effluent fractions containing each isomer were collected for further investigation. The conformational states of the peptide isomers upon separation were conserved by storing the effluent fractions in liquid nitrogen. The Leu-Pro, Phe-Pro, Tyr-Pro and Tyr-Pro-Phe conformers were identified by one- and two-dimensional NMR spectroscopy at −15°C. Upon comparing the NMR spectra of the isomers, for these peptides the retention order of the conformers was unambiguously established: in each case the trans conformer is eluted before the cis conformer. On the basis of NMR data obtained with β-casomorphin-5, which contains two proline residues, the elution order of its four conformers was established by NMR spectroscopy of the fractions obtained by RP-HPLC at low temperature as trans-trans (least retained), trans-cis, cis-cis and cis-trans (most retained).