IR and Raman characterization of carcinine tautomers as a function of pH and copper (II) complex formation.

Carcinine (p-alanyl-histamine) (Care) is a natural imidazole-containing compound found in the non-protein fraction of mammalian tissues and probably involved in the carnosine-histidine-histamine metabolic pathway in physiologic response to stress. It has a good potential to act as a natural antioxidant in vivo since it is able to scavenge harmful "OH radicals and inhibit fatty acid peroxidation. One of the mechanism by which antioxidants protects targets from oxidative stress is the chelation of transition metals, such as copper, preventing them from participating in deleterious processes. The ability of Care to chelate copper ions was tested by a vibrational study of Care alone and its 1:1 complexes with Cu(II) ions at different pHs (7, 9 and 10). Both Raman and IR spectra present marker bands useful for analyzing the tautomeric equilibrium of the imidazole ring and identifying the complex structure predominating at a specific pH. Tautomer I (NT-H) is the preferred form of free Care both at neutral and basic pH (Raman and IR bands at ~ 1578, 1292 and 988 cm-1). In addition, free Care is able to form a strong network of H-bonds by its three differently protonated nitrogen atoms (-NHs+, peptidic -NH- and imidazolic >NH) and the peptidic C=O and/or the non-protonated N imidazolic atom. With regards the metal interaction, Care is a polydentate ligand offering four potential binding sites: the two imidazole nitrogen, the amino group and the peptide linkage. The presence of copper ions does not strongly affect the tautomerie equilibrium at neutral pH. The Raman bands of the imidazolic ring were observed at different wavenumbers (1596 and 972 cm-1) respect to free Care, indicating that tautomer I is involved in the coordination. By increasing pH, the deprotonation of both N-imidazolic nitrogens is induced, giving rise to metal imidazolate bridges. Both at neutral and basic pH the primary amine group takes part to the Cu chelation, whereas only under neutral conditions all the peptidic moieties are involved in coordination. Thus, Care acts as a tri-dentate ligand at neutral pH, mainly giving a monomeric complex, [CuLH-1], containing tautomer I. By increasing pH up to 10, an oligonuclear complex, probably [Cu4L4H4], with metal-imidazolate ions connecting different ligand molecules predominates.