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 (beta-casomorphin 1-3 fragment),
Tyr-Tic-
Phe-Phe, Try-
Pro-Phe-Pro-Gly (beta-casomorphin-5), Tyr-Tic-Phe-Phe-Val-Val-Gly-NH2 and Tyr-Tic-Phe-Gly-Tyr-Pro-Ser-NH2. 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 degrees 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 the beta-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).