Chiara Conato

Copper complexes of glycyl-histidyl-lysine and two of its synthetic analogues: chemical behaviour and biological activity.

Copper complex formation equilibria of glycyl-L-histidyl-L-lysine (Gly-His-Lys, GHK) and of two synthetic analogues, where the histidine residue was replaced with a synthetic amino acid (L-spinacine or L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid), have been carefully investigated using different experimental techniques: potentiometry, solution calorimetry, UV-VIS spectrophotometry, circular dichroism and electron paramagnetic resonance spectroscopies. All the ligands formed complexes having different stoichiometries and stabilities; evidence for the formation of binuclear species is also shown. The structures of the main complexes are discussed. It is suggested that the lateral lysine amino group participates in complex formation, but only at alkaline pH values: at physiological pH this group is protonated and available for possible interactions with cellular receptors. The above tripeptides have been tested for their enzymatic stability in human serum: the synthetic compounds showed no significant degradation for at least 3 h. Finally, their activity as growth factor has been studied in vitro. The two synthetic analogues showed an activity comparable to or even higher than that of GHK, thus suggesting their possible use as additives in cell culture media, even in the presence of serum. Relevant information on the GHK action mechanism as cell growth factor has been obtained: the formation of copper complexes, driven by the first (Gly) residue, appears necessary while the second residue (His) does not appear to play a specific role; the presence of the free side chain of the third residue (Lys) appears to be of fundamental importance.

Copper(II) complexes with l-lysine and l-ornithine: is the side-chain involved in the coordination?: A thermodynamic and spectroscopic study

Abstract The involvement of the ω-NH 2 terminal group of l -lysine and l -ornithine in the complexation of Cu(II) was investigated by means of potentiometry, calorimetry, UV–Vis and ESR. The thermodynamic parameters (Δ G 0 , Δ H 0 and Δ S 0 ) obtained by combining the potentiometric and calorimetric data show that the two ligands complex copper(II) in a different manner, indicating that the ω-NH 2 terminal group is involved in ornithine complexes only. UV–Vis and ESR spectra further support the involvement of ornithine ω-NH 2 and provide details on the complexation geometries of the complexes of the two amino acids.

Copper and nickel complex-formation equilibria with Lys/Gly/His/ Lys, a fragment of the matricellular protein SPARC

Complex-formation equilibria of the tetrapeptide Lys/Gly/His/Lys with the Cu(II) and the Ni(II) ions have been studied in aqueous solution, at I/0.1 mol dm 3 (KNO3) and T/25 8C. Protonation and complex-formation constants have been potentiometrically determined. The structure of the main complex species is discussed on the basis of thermodynamic data obtained by direct calorimetry as well as the CD, ESR and electronic spectra. The participation of two amide nitrogens in complex-formation is suggested for both the metal ions, while no evidence supports the participation of the o-NH2 side groups of Lys residues in coordination. # 2002 Elsevier Science Ltd. All rights reserved.

Experimental approaches for size-based metal speciation in riversElectronic supplementary information (ESI) available: Experimental details, tables A?D showing parameters for GF and AAS, detection limits for ETAAS, SdFFF operative parameters and metal concentrations determined in the void time at the two different pH values, and SEM images of the void time fractions. See http://www.rsc.org/suppdata/em/b3/b308877d/

A review of the different methodologies employed to fractionate and characterize riverine suspended particulate matter is presented. The importance of size-based metal speciation is underlined and the possibility of studying it by the Sedimentation Field Flow Fractionation (SdFFF) technique is illustrated. The studies on the metal load in river Suspended Particulate Matter (SPM) performed over the last ten years are critically reviewed focusing on the different methods employed to collect, concentrate and size-fractionate samples. The fact that there is no homogeneity in methods and data collection in this field is underlined. Among the different fractionation techniques, Field Flow Fractionation (FFF) methodologies have proved to be a good approach to study the role of SPM in metal load and transport. The possibility of studying size-based metal speciation using the SdFFF technique is presented and the importance of metal speciation in rivers is underlined.

Copper complexes of dipeptides with l-Lys as C-terminal residue: a thermodynamic and spectroscopic study

Abstract Complex-formation equilibria of some dipeptides (glycyl-lysine, alanyl-lysine, histidyl-lysine and β-alanyl-lysine) with the Cu(II) ion have been studied in aqueous solution, at I =0.1 moldm −3 (KNO 3 ) and T =25°C. Protonation and complex-formation constants have been determined potentiometrically; complex-formation model and species stoichiometry have been checked carefully by means of different spectroscopic techniques (visible absorption, CD, EPR). The structure of the main complex species is discussed, on the basis of thermodynamic data obtained by direct calorimetry. The formation of binuclear species is demonstrated and it is suggested that the side amino group of lysine participates in complex formation at alkaline pH values.

Cu(II) ion coordination to SPARC: a model study on short peptide fragments

SPARC (secreted protein, acidic and rich in cysteine) is a glycoprotein of the extracellular matrix that mediates the cell-matrix interactions. It plays also a role in angiogenesis, tumorigenesis, caractogenesis and wound healing. The human SPARC consists of three distinct modules. Module II is follistatin-like and its hydrolysis gives rise to a number of oligopeptides that can regulate angiogenesis in vivo and the biological activity of which has been related to their association with endogenous or exogenous copper ion. In order to completely understand the biological role of metal complexes formed by SPARC and its fragments, more information is needed on their stoichiometry, stability and structure in solution. In the present paper a potentiometric and spectroscopic investigation on Cu(II) complexes with the three SPARC122–126, SPARC121–126 and SPARC120–126 fragments, protected at both their amino and carboxylic ends, is reported. These peptides (Ac-HKLHL-NH2, Ac-GHKLHL-NH2 and Ac-KGHKLHL-NH2, respectively) constitute good models for the strong copper-binding site of the protein. The behaviour of the three ligands is very similar: complex formation is started by the two His residues, subsequently involving up to three amido nitrogens, as pH increases. The coordination of the two histydyl imidazoles promotes amide ionization in the physiological pH range and this can explain SPARC binding to the Cu(II) ion.

Direct chiral resolution of underivatized amino acids on a stationary phase dynamically modified with the ion-exchanger Nτ-decyl-l-spinacine

Increasing attention has been devoted in the last decades to chiral chromatography, principally to high-performance liquid chromatography techniques using a chiral stationary phase. Many chiral high-performance liquid chromatography columns are commercially available, but, unfortunately, they are most often rather expensive. A cheap alternative to the commercial chiral columns is the dynamic-coating procedure of a standard achiral stationary phase with a chiral selector containing both a chiral domain and a chain or a group able to tightly (but noncovalently) bind the achiral support. This is the case of N(τ) -decyl-l-spinacine, already successfully employed to dynamically cover a reversed-phase column to separate racemic mixtures of amino acids through the ligand-exchange mechanism. In the present work, the same chiral selector is employed to separate racemic mixtures of amino acids and oligopeptides, in the absence of metal ions: no coordination complex is formed, but only electrostatic and weak nonbonding interactions between the chiral phase and the analytes are responsible for the observed enantioselectivity. The new method is simpler than the previous one, very effective in the case of aromatic amino acids and oligopeptides and also suitable for preparative purposes.

Chiral ligand-exchange resolution of underivatized amino acids on a dynamically modified stationary phase for RP-HPTLC.

The synthesis of Spi(τ-dec), derived from the selective alkylation of L-spinacine (4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid) at the τ-nitrogen of its heteroaromatic ring, with a linear hydrocarbon chain of 10 carbon atoms, is described here for the first time. Spi(τ-dec) was successfully employed in the past to prepare home-made chiral columns for chiral ligand-exchange high-performance liquid chromatography. In the present article a new method is described, using Spi(τ-dec) as a chiral selector in high-performance thin-layer chromatography (HPTLC): commercial hydrophobic plates were first coated with Spi(τ-dec) and then treated with copper sulfate. The performance of this new chiral stationary phase was tested against racemic mixtures of aromatic amino acids, after appropriate optimization of both the conditions of preparation of the plates and the mobile phase composition. The enantioselectivity values obtained for the studied compounds were higher than those reported in the literature for similar systems. The method employed here for the preparation of chiral HPTLC plates proved practical, efficient, and inexpensive.

Cu(II) ion coordination to the pentadecapeptide model of the SPARC copper-binding site

SPARC (Secreted Protein, Acidic and Rich in Cysteine) is a matricellular glycoprotein with many biological functions: it mediates the interactions between cells and the extracellular matrix, playing a role in angiogenesis, tumorigenesis, caractogenesis and wound healing. Proteolysis of SPARC gives rise to a number of oligopeptides which can regulate angiogenesis in vivo and the biological activity of which has been related to their association with endogenous or exogenous copper ion. Human SPARC consists of three distinct modules. Module II is follistatin-like and contains two copper binding sites, the strongest of which—the cationic region 2 (amino acids 114–130)—contains the sequence Gly–His–Lys. In order to shed more light on the biological role of metal complexes formed by SPARC and its fragments, more information is needed on their stoichiometry, stability and structure in solution. In the present paper a potentiometric and spectroscopic investigation on Cu(II) complexes with the SPARC114–128 fragment, protected at both its amino and carboxylic ends, is reported. This peptide (Ac–TLEGTKKGHKLHLDY–NH2) constitutes a good model to the strong copper-binding site of the protein. The whole experimental data suggest that complex-formation is started by the two His residues, subsequently involving up to three amido nitrogens, as pH increases. The coordination of the two histydyl imidazoles is able to promote amide ionisation in the physiological pH range and this could be the key to the SPARC affinity for Cu(II) ion.