RESUMEN
The solution properties of a series of (pyridin-2-yl)aminomethane-1,1-diphosphonic acids 1-4 as well as closely related compounds 5 and 6 with 1,3-thiazolyl or 1,3-benzothiazolyl side chains and their influence on the complexation of Zn(II), Mg(II) and Ca(II) were studied by means of the 31P and 1H NMR spectroscopy, pH-potentiometry and ESI-MS methods. The crystal structures of 5 and 6 were determined by X-ray analysis. All the studied compounds exist in solution as mixtures of the Z and E isomers with respect to the C2-Namino bond. Compounds 1 and 2 exhibit higher overall basicities compared to 3-6 and have exceptionally basic pyridyl nitrogen (pK2 = 7.88 and 8.18, respectively). Dynamic NMR studies revealed that the nature of the aromatic side chain as well as the specificity and topology of the ring substituent account for the rotational barrier for the Z/E interconversion, which decreases in the order of 1 approximately 2 > 3 approximately 4 >> 5 (6). This is particularly important for aggregational properties of 1-6 in solution and for the complex-formation equilibria. Overall, compounds 1-6 demonstrate a strong tendency for the formation of protonated multinuclear complexes. Their formation is unexpectedly slow on the NMR time scale compared to previously studied bisphosphonate complexes. The stepwise release of protons upon the rise of pH leads to mononuclear species. The Z ligand conformation is preferred in multinuclear complexes of Zn(II), Mg(II) and Ca(II) with 1 and 2 whereas in the case of 3-6 the complexation processes are affected by the intramolecular Z/E interconversion.
Asunto(s)
Calcio/química , Complejos de Coordinación/química , Difosfonatos/química , Magnesio/química , Zinc/química , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Conformación Molecular , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Metal-catalyzed oxidation (MCO) of proteins is mainly a site-specific process in which one or a few amino acids at metal-binding sites on the protein are preferentially oxidized. The oxidation of proteins by MCO can lead to oxidation of amino acid residue side chains, cleavage of the peptide bonds and formation of covalent protein-protein cross-linked derivatives. In an attempt to elucidate the products of the copper(II)-catalyzed oxidation of the 29-56, M29-D30-56 and Ac-M29-D30-56 fragments of alpha-synuclein, high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) methods and Cu(II)/hydrogen peroxide as a model oxidizing system were employed. The peptide solution (0.50 mM) was incubated at 37 degrees C for 24 h with metal : peptide : hydrogen peroxide 1 : 1 : 4 molar ratio in phosphate buffer, pH 7.4. Oxidation targets for all studied peptides are the histidine residues coordinated to the metal ions. For the M29-D30-56 and Ac-M29-D30-56 peptides the oxidation of the methionine residue to methionine sulfoxide and sulfone is observed. The cleavage of the peptide bond M29-D30 for the M29-D30-56 peptide was detected as metal binding residues. The fragmentations of the M29-D30-56 peptide near the Lys residues were observed supporting the participation of this (Lys) residue in the coordination of the copper(II) ions.
Asunto(s)
Cobre/metabolismo , Peróxido de Hidrógeno/metabolismo , Fragmentos de Péptidos/metabolismo , alfa-Sinucleína/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Cationes Bivalentes , Cromatografía Líquida de Alta Presión , Cobre/química , Histidina/química , Histidina/metabolismo , Peróxido de Hidrógeno/química , Concentración de Iones de Hidrógeno , Lisina/química , Lisina/metabolismo , Metionina/análogos & derivados , Metionina/química , Metionina/metabolismo , Datos de Secuencia Molecular , Oxidación-Reducción , Fragmentos de Péptidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Sulfonas/química , Sulfonas/metabolismo , alfa-Sinucleína/químicaRESUMEN
Stability constants and ligand donor sets of the copper(II) complexes of the NH2-29-56(L1)(AA30GKTKEGVLYV40GSKTKEGVVH50GVATVA56-NH2), NH2-M29-D30-56(L2) and Ac-M29-D30-56(L3) fragments of alpha-synuclein were determined in aqueous solution for 1 : 1 metal-to-ligand molar ratio in the pH range 2.5-10.5. The tyrosine residue in the 39th position of the alpha-synuclein fragments does not take part in the coordination of the metal ion. The potentiometric and spectroscopic data (UV-Vis, CD, EPR) show that acetylation of the amino terminal group induces significant changes in the coordination properties of the L3 fragment compared to that of the L2 peptide. When the amino group is blocked (L3) the imidazole nitrogen of the histidine residue acts as an anchoring site and at higher pH the 3N {N(Im),2N-} and 4N {N(Im),3N-} complexes are formed. The L1 peptide at physiological pH forms in equilibrium 3N {NH2,N-,CO,N(Im)} and 4N {NH2,2N-,N(Im)} complexes. For the L2 peptide the coordination of the copper(II) ions starts from the N-terminal Met residue and with increasing of pH the Asp residue in second position of amino acid sequence coordinates and stabilizes significantly the 2N complex as a result of chelation through the beta-carboxylate group. At physiological pH the 3N {NH2,N-,beta-COO-,N(Im)} coordination mode dominates. At pH above 6 the results for the L2 fragment suggest the formation of 3N and 4N complexes (in equatorial plane) and the involvement of the lateral NH2 group of Lys residue in the axial coordination of Cu(II) ion. In CD spectra sigma (epsilon-NH2-Lys) --> Cu(II) charge transfer transition is observed. The stability constants for the L2 fragment of alpha-synuclein of the 4N {NH2,2N-,N(Im)} and {NH2,3N-} complexes are higher about 1.5 and 0.7 orders of magnitude, respectively, by comparison to those of the L1 peptide. This increase may be explained by the involvement of the epsilon-NH2 group of Lys residue in the coordination sphere of metal ion.
Asunto(s)
Cobre/metabolismo , Fragmentos de Péptidos/química , alfa-Sinucleína/metabolismo , Aminoácidos , Sitios de Unión , Humanos , Potenciometría , Unión Proteica , Análisis Espectral , alfa-Sinucleína/químicaRESUMEN
Reactive oxygen species (ROS) may provide the covalent modifications of amino acid residues in proteins, formation of protein-protein cross-linkages, and oxidation of the protein backbone resulting in protein fragmentation. In an attempt to elucidate the products of the copper(II)-catalyzed oxidation of the (1-17), (1-28), (1-39) and (1-39)(A30P) fragments of alpha-synuclein, the high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) methods and Cu(II) /hydrogen peroxide as a model oxidizing system were employed. Peptide solution (0.50 mM) was incubated at 37 degrees C for 24 h with metal:peptide:hydrogen peroxide molar ratio 1:1:4 in phosphate buffer, pH 7.4. Oxidation targets for all peptide studied are the methionine residues (M(1), M(5)). Incubation 24 h of the (1-28), (1-39) and (1-39)(A30P) fragments in aerobic conditions lead to the oxidation of one methionine residue to methionine sulfoxide. Reaction of hydrogen peroxide with all fragments of alpha-synuclein resulted in oxidation of two methionine residues (M(1), M(5)) to methionine sulfoxides. For the Cu(II):peptide:hydrogen peroxide 1:1:4 molar ratio systems the further oxidation of methionine residues to sulfone was observed. The cleavage of the peptide bond M(1)-D(2) for all peptides studied was observed as metal binding residues. For the (1-39) and (1-39)(A30P) fragments of alpha-synuclein the molecular ions with lower molecular masses (A(11)-Y(39), E(13)-Y(39)) were also detected.
Asunto(s)
Cobre/metabolismo , Peróxido de Hidrógeno/metabolismo , alfa-Sinucleína/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cromatografía Líquida de Alta Presión/métodos , Peróxido de Hidrógeno/química , Metionina/química , Metionina/metabolismo , Datos de Secuencia Molecular , Oxidación-Reducción , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , alfa-Sinucleína/químicaRESUMEN
Copper(II) complexes of the 1-17 (MDVFMKGLSKAKEGVVA-NH(2)), 1-28 (MDVFMKGLSKAKEGVVAAAEKTKQGVAE-NH(2)), 1-39 (MDVFMKGLSKAKEGVVAAAEKTKQGVAEAPGKTKEGVLY-NH(2)) and 1-39 (A30P) fragments of alpha-synuclein were studied by potentiometric, UV-Vis (UV-visible), CD (circular dichroism) and EPR (electron paramagnetic resonance) spectroscopic methods to determine the stoichiometry, stability constants and coordination modes of the complexes formed. The beta-carboxylate group of Asp residue in second position of the peptide chain coordinates strongly to Cu(II) ion over the pH range 4-9.5 to give unusually stable 2N complex with {NH(2), N(-), beta-COO(-), H(2)O} coordination mode. At pH above 7 the results suggest the formation of 2N, 3N, 4N complexes (in equatorial plane) and the involvement of the lateral NH(2) group of Lys residue in the axial coordination of Cu(II) ion. In CD spectra sigma (epsilon-NH(2)-Lys)-->Cu(II) charge transfer transition is observed. Addition of the 18-28 and 18-39 fragments to the 1-17 peptide does not change the coordination mode and the 1-39 fragment forms the Cu(II) complexes with higher stabilities compared to those of the 1-17, 1-28 and 1-39(A30P) fragments of alpha-synuclein.