β-Casomorphin (1-3)

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).

An X-prolyl-dipeptidyl peptidase has been purified from Lactobacillus sakei by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55 degrees C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu(2+), Hg(2+), and Zn(2+)). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates. K(m) s for Gly-Pro- and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 microM, respectively; those for Gly-Pro- and Ala-Pro-p-nitroanilide were 192 and 50 microM, respectively. Among peptides, beta-casomorphin 1-3 was hydrolyzed at the highest rates, while the relative hydrolysis of the other tested peptides was only 1 to 12%. The potential role of the purified enzyme in the proteolytic pathway by catalyzing the hydrolysis of peptide bonds involving proline is discussed.

The tetrapeptide Boc-D-Orn-Phe-D-Pro-Gly-OH and the pentapeptide sequence Boc-Tyr(tBu)-D-Orn-Phe-D-Pro-Gly-OH were used to study the influence of different coupling reagents on the yield and purity of these model peptides. The simple structure prevented racemization and cyclodimerization and facilitated the ring formation. The most favorable effects on yield and purity were obtained in both reactions using diphenyl-phosphoryl azide (DPPA) and norborn-5-ene-2,3-dicarboximidodiphenylphosphate (NDPP), while the cyclizations with the powerful activating reagents benzotriazol-1-yl-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate (BOP) and 2-(1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) with the exception of the cyclopentapeptide reaction with HBTU/4-dimethylaminopyridine gave unsatisfactory results.