The formation of imidazolines within the backbone during heating of peptides and proteins was studied. In model experiments, the generation of 2-methyl-4-carboxy-imidazoline after incubation of N-alpha-stop-acetyl-beta-aminoalanine was demonstrated by cation-exchange chromatography and NMR-studies. At 60 degrees C the highest concentrations of this imidazoline were measured at pH 9. With increasing temperature the pH-optimum of the imidazoline formation shifted to lower pH levels.

The reaction of N-alpha-acetyl-dehydroalanine-methylester with ammonia generated beta-aminoalaninoalanine, which was identified in comparison with synthesized references by HMQC-and(1)H-NMR-spectroscopy. beta-Aminoalaninoalanine, a crosslink-product homologous to lysinoalanine, which reacts easily with the corresponding imidazoline. Thus, the formation of imidazolines in peptide backbones containing beta-aminoalanine was shown unequivocally.

Further studies were conducted with peptides, synthesized according to the 10-28 sequence of bovine beta-caseine with beta-aminoalanine respectively beta-alanine substituted for a phosphoserine residue. After incubation, the loss of one molecule of water from the beta-aminoalanine containing peptide, but not from the native peptide or the peptide containing beta-alanine, was detected by HPLC and MS. As a consequence, the loss of water was explained by cyclization and the formation of an imidazoline.

These investigations indicate that the formation of imidazolines-besides the formation of thiazolines-takes place during heating of peptides and proteins. Heterocyclic backbone-modifications are a form of posttranslational modifications, which may play a role during the processing of proteins and subsequent formation of unexpected compounds.