ISOLATION AND IDENTIFICATION OF PEPTIDE CONFORMERS BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND NMR AT LOW-TEMPERATURE

Peptide conformers with one or more rotationally hindered peptide bond s due to the presence of proline and/or another N-substituted amino ac id residue in the molecule were separated by reversed-phase chromatogr aphy at low temperatures, isolated and identified by NMR. The scope of this investigation included the cis-trans isomers of the dipeptides L eu-Pro, Phe-Pro and Tyr-Pro as well as conformers of opioid peptides c ontaining proline and/or the proline-like Tic (1,2,3,4-tetrahydro-isoq uinoline-3-carboxylic acid) residues: Tyr-Pro-Phe (beta-casomorphin 1- 3 fragment), Tyr-Tic-Phe-Phe, Tyr-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 e ffluent fractions containing each isomer were collected for further in vestigation. The conformational states of the peptide isomers upon sep aration were conserved by storing the effluent fractions in liquid nit rogen. The Leu-Pro, Phe-Pro, Tyr-Pro and Tyr-Pro-Phe conformers were i dentified by one- and two-dimensional NMR spectroscopy at -15 degrees C. Upon comparing the NMR spectra of the isomers, for these peptides t he 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 beta-casomorphin-5, which contains two proline residues, the elution order of its four conformers was es tablished by NMR spectroscopy of the fractions obtained by RP-HPLC at low temperature as trans-trans (least retained), trans-cis, cis-cis an d cis-trans (most retained).