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A former spectroscopic study of Cu(II) coordination by the 13-membered ring cyclic tetrapeptide c(Lys-dHis-βAla-His) (DK13), revealed the presence, at alkaline pH, of a stable peptide/Cu(III) complex formed in solution by atmospheric dioxygen oxidation. To understand the nature of this coordination compound and to investigate the role of the His residues in the Cu(III) species formation, Cu K-edge XANES, and EXAFS spectra have been collected for DK13 and two other 13-membered cyclo-peptides: the diastereoisomer c(Lys-His-βAla-His) (LK13), and c(Gly-βAla-Gly-Lys) (GK13), devoid of His residues. Comparison of pre-edge peak features with those of Cu model compounds, allowed us to get information on copper oxidation state in two of the three peptides, DK13 and GK13: DK13 contains only Cu(III) ions in the experimental conditions, while GK13 binds only with Cu(II). For LK13/Cu complex, EXAFS spectrum suggested and UV-vis analysis confirmed the presence of a mixture of Cu(II) and Cu(III) coordinated species. Theoretical XANES spectra have been calculated by means of the MXAN code. The good agreement between theoretical and experimental XANES data collected for DK13, suggests that the refined structure, at least in the first coordination shell around Cu, is a good approximation of the DK13/Cu(III) coordination species present at strongly alkaline pH. All the data are consistent with a slightly distorted pyramidal CuN(4) unit, coming from the peptide bonds. Surprisingly, the His side-chains seemed not involved in the final, stable, Cu(III) scaffold.

The tetrapeptide Ac-Gly-[Cys-Sec]-Gly-NH(2), reproducing the C-terminal motif of the selenoenzyme thioredoxin reductase, was designed and synthesized, and its reactions with a few medicinally relevant gold(i,iii) compounds investigated by ESI-MS. Remarkably, the main reaction products could be unambiguously identified providing valuable insight into the likely mechanisms of enzyme inhibition by gold compounds.

A series of linear tetrapeptides containing two histidyl residues in position 2 and 4, namely DHGH, DHGdH, KHGH, KHGdH, Ac-DHGH-NH(2), Ac-DHGdH-NH(2), Ac-KHGH-NH(2), and Ac-KHGdH-NH(2), were synthesized and characterised. Their copper(II) binding properties were investigated in depth through a variety of physicochemical methods. Potentiometric titrations were first carried out to establish the stoichiometry and the stability of the resulting copper(II)-peptide complexes. The copper(II) chromophores that are formed in the various cases in dependence of pH were subsequently characterised by extensive spectroscopic analysis (UV-Vis, EPR, CD) in strict correlation with potentiometric data. The effects of the nature of the first amino acid (Lys versus Asp) and of N-terminal amino group protection on copper(II) binding were specifically addressed. On turn, the careful comparison of the copper(II) coordination abilities of the linear peptides with those of their cyclic analogs provided insight into the effects of cyclization on the overall metal binding properties.

We describe synthesis, conformational studies, and binding to the five somatostatin receptors (sst 1-5) of a few analogues of the cyclic octapeptide octreotide (1), where the disulfide bridge was replaced by a dicarba group. These analogues were prepared by on-resin RCM of linear hepta-peptides containing two allylglycine residues; first- and second-generation Grubbs catalyst efficiencies were compared. The C=C bridge was hydrogenated via two different methods. Binding experiments showed that two analogues had good affinity and high selectivity for the sst5 receptor. Three-dimensional structures of the active analogues were determined by (1)H NMR spectroscopy. Conformation-affinity relationships confirmed the importance of D-Phe(2) orientation for sst2 affinity. Moreover, helical propensities well correlates with the peptide sst5 affinity. The presence of the bulky aromatic side chain of Tyr(Bzl)(10) favored the formation of a 3(10)-helix and enhanced the sst5 selectivity suppressing the sst2 affinity. Finally, a new pharmacophore model for the sst5 was developed.

Cyclotetrapeptides are constrained cyclic peptides whose synthesis is considered a difficult task. A methodology based on on-resin head-to-tail cyclization by anchoring the side chain of a trifunctional amino acid was investigated. A series of model cyclotetrapeptides containing the RGD sequence cyclo(Xaa-Arg-Gly-Asp) (Xaa = Ala, Phe, Phg, D-Ala, D-Phe, D-Phg) was synthesized with no cyclodimerization by-products. An evaluation and optimization study of all of the parameters directly involved in the ring closure was performed.

Stoichiometry, stability constants and solution structures of the copper(II) complexes of the N-acetylated tetrapeptide HisGlyHisGly were determined in aqueous solution in the pH range 2-11. The potentiometric and spectroscopic data (UV-Vis, CD, EPR and Raman scattering) show that acetylation of the amino terminal group induces drastic changes in the coordination properties of AcHGHG compared to HGHG. The N3 atoms of the histidine side chains are the first anchoring sites of the copper(II) ion. At pH 4.7 and 5.6 both the imidazole rings cooperate in the formation of a 2N equatorial set, while, at higher pH values, 3N and 4N complexes are formed through the coordination of peptide N- atoms. The logbeta values of the copper complexes of AcHGHG are by far lower than those of the corresponding species in the parent CuII-HGHG system.

The tetrapeptide-Cu(II) complex H-(l-His-Gly)2-OH/Cu(II), indicated as L-Cu(II), has been investigated, as compared to the Cu(II) inorganic salt CuSO4, for its antioxidative and anti-inflammatory properties under a panel of experimental conditions. Both inorganic and organic Cu(II) compounds showed comparable activities in vitro and ex vivo by: (i) protecting, in a dose-dependent manner, rat brain homogenates from Fe(III)/ascorbate- or haemoglobin-induced lipid peroxidation; (ii) inhibiting the superoxide-mediated ferricytochrome c reduction by activated macrophages. CuSO4 and L-Cu(II) also exhibited similar anti-inflammatory effects in vivo by reducing significantly the extent of carrageenan-induced edema in the rat paw. The activities of the two compounds diverged strikingly only in the xanthine/xanthine oxidase system at low phosphate buffer concentration. L-Cu(II) decreased the rate of NBT reduction by superoxide in a true SOD-like fashion without affecting urate production. Instead, Cu(II) ions caused the rapid xanthine oxidase inactivation thus inhibiting both urate and superoxide production; this effect might be ascribed to the superoxide-mediated generation of the strong oxidant Cu(III) and its interaction with the enzyme. The administration of Cu(II), whether complexed with linear oligopeptides or as an inorganic salt, to animals or tissue extracts, conferred protection against oxidation and ought, conceivably, to interact with endogenous biological molecules and form highly bioavailable complexes which serve, subsequently, as the real scavengers. Moreover, the claimed prominent scavenger activities of Cu(II)-oligopeptide complexes over inorganic copper ions could be realised only in very simple in vitro systems through mechanisms which, although of biochemical interest, are unlikely to be of physiopathological significance., (Copyright 1998 The Italian Pharmacological Society)