1H NMR studies of natural and synthetic immunostimulating peptides in aqueous solution

The peptides LAla-DGlu (L-alanyl-D-glutamic acid), LAla-DGlu[LLA2pm(Gly)]NH2, i.e. N2-(L-alanyl-D-gamma-isoglutaminyl)-N6-glycyl-LL-2,6-diaminopimelic acid, and LAla-DGlu[LLA2pm(Gly)-D-Ala], i.e. N2-(L-alanyl-D-gamma-glutamyl)-N6-glycyl-LL-alpha-2,6-diaminopimelyl-D-alanine, extracted from a Streptomyces strain and their immunostimulating synthetic lauroyl derivatives were studied by 1H NMR spectroscopy at 270 MHz. The chemical shift analysis confirms (a) the sequence of these compounds, (b) the occurrence of a gamma bond between Glu (or GluNH2) and A2pm, (c) the presence of a carboxamide group on the backbone of the tetrapeptide LAla-DGlu[LLA2pm(Gly)]. Temperature and pH studies strongly suggest that the tetrapeptide and pentapeptide exist under several folded conformations characterized by a proximity between the NH3 group of Gly and the Glu (or GluNH2) residue. THe similarity between the chemical shifts of the corresponding protons in LAla-DGlu, lauroly-LAla-DGlu and muramyl-L:Ala-DGluNH2 shows that the coupling of the dipeptide with a lauroyl or a muramyl group does not induce significant changes in the structure of the peptide moiety. In the lipopeptide series the presence of the lauroyl chain leads to amphipathic substances which form, micelles in aqueous solution as shown by the large increase of the proton linewidths. The immunostimulating properties of this kind of peptide manifest themselves at very low concentration and only after their coupling with a sugar or lipid moiety. These features can be interpreted by the formation of mixed complexes between the immunoadjuvants and cell membrane constituents.