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Gold-based drugs typically behave as strong inhibitors of the enzyme thioredoxin reductase (hTrxR), possibly as the consequence of direct Gold(I) coordination to its active site selenocysteine. To gain a deeper insight into the molecular basis of enzyme inhibition and prove gold-selenocysteine coordination, the reactions of three parent Gold(I) NHC compounds with the synthetic C-terminal dodecapeptide of hTrxR containing Selenocysteine at position 498, were investigated by electrospray ionization mass spectrometry (ESI-MS). Formation of 1:1 Gold-peptide adducts, though in highly different amounts, was demonstrated in all cases. In these adducts the same [Au-NHC](+) moiety is always associated to the intact peptide. Afterward, tandem MS experiments, conducted on a specific Gold-peptide complex, pointed out that Gold is coordinated to the selenolate group. The relatively large strength of the Gold-selenolate coordinative bond well accounts for potent enzyme inhibition typically afforded by these Gold(I) compounds. In a selected case, the time course of enzyme inhibition was explored. Interestingly, enzyme inhibition turned out to show up very quickly and reached its maximum just few minutes after mixing. Overall, the present results offer some clear insight into the process of thioredoxin reductase inhibition by Gold-based compounds., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A new, 14-membered, tetraza cyclic tetrapeptide containing histidine and lysine side-chains, c(beta(3)homoLysdHisbeta-AlaHis), was designed, synthesized and characterized; its copper(II) binding properties were investigated in dependence of pH by potentiometric and spectroscopic methods. In line with previous studies of similar systems, the progressive involvement of amide nitrogens in copper(II) coordination was evidenced for pH values greater than 6. At physiological pH the dominant species consists of a copper(II) center coordinated by two amide nitrogens, an imidazole nitrogen and a water molecule. In contrast, at pH values higher than 8.7, a copper(II) coordination environment consisting of four amide nitrogens in the equatorial plane and the axial imidazole ligands is formed as clearly indicated by spectroscopic data and theoretical calculations. The behavior of this 14-membered cyclic tetrapeptide is compared to that of its 12-membered cyclic analog, particular attention being paid to the effects of ring size on the respective copper(II) binding abilities.

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.

Three novel cyclic tetrapeptides, containing either l- or d-histidine residues and either Lys or Asp side chains, namely c(HGd-HK) (1), cHGHD (2) and c(HGd-HD) (3), were designed, synthesized, characterized and tested as potential copper(II) ligands. Their pH dependent copper(II) binding properties were analysed in depth by a number of potentiometric and spectroscopic determinations. A rather exhaustive description of the species existing in solution has emerged for each copper(II)/oligopeptide system; solution structures for the individual species are proposed. The specific role of the various side chains in the overall metal coordination process is discussed in comparison with the case of Cu(II)-c(HGHK), previously reported. Data obtained in this study highlight the strong impact of the d-His residue on the metal binding abilities of these cyclic peptides. Remarkably, the cyclic tetrapeptides containing two l-His residues are able to form, at physiologically relevant pH values, a characteristic chromophore where the mononuclear copper(II) centre is simultaneously coordinated by two imidazole nitrogens and two amidic nitrogens of the tetrazadodecane ring. This latter type of copper(II) chromophore has been carefully modelled by computational methods. The potentialities of the applied experimental strategy are stressed.

The new cyclic tetrapeptide c(HGHK) was synthesised in the solid phase and its complexes with copper(II) were studied in aqueous solution at various pH values by means of potentiometric and spectroscopic methods (UV, EPR, CD). Six mononuclear coordination species were clearly identified within the pH range 3-11. Spectroscopic data strongly suggest sequential formation of N, 2N, 3N and 4N equatorial donor sets around the copper(II) centre from the lowest to the highest pH, involving both imidazole nitrogens and amide nitrogens. A detailed comparison with the copper(II) binding properties of HGHG and Ac-HGHG ligands is also reported.

The complexes between copper(II) and four synthetic tetrapeptides bearing a single histidine residue within the sequence (AcHGGG, AcGHGG, AcGGHG and AcGGGH, respectively), have been investigated by potentiometric and spectroscopic methods (UV-Vis, circular dichroism and electron paramagnetic resonance). Potentiometric studies in the pH range 4-12 allowed identification and quantitative determination of the species present in solution for each copper-peptide complex. In all cases, upon raising pH, copper(II) coordination starts from the imidazole nitrogen of the His; afterwards three deprotonated amide nitrogens are progressively involved in copper coordination, except in the case of AcGHGG. Based on the potentiometric and spectroscopic results, detailed molecular structures are proposed for the dominant copper(II) tetrapeptide species existing in solution, either at neutral or alkaline pH. The structural consequences of the presence and of the location of a unique histidine residue within the tetrameric sequence are specifically analyzed. Results are discussed in relation to the modeling of copper(II) binding sites in proteins, particular emphasis being devoted to the copper complexes of the prion protein.

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.