Copper(II)-lincomycin: complexation pattern and oxidative activity

Coordination of Cu(II) to lincomycin was studied by potentiometry, UV-Vis, circular dichroism (CD), EPR, NMR, cyclic voltammetry (CV) and ESI-MS. Only mononuclear complexes of stoichiometries ranging from CuL to CuH(-3)L were found. In the main species present at neutral pH, CuH(-2)L, lincomycin bonds Cu(II) through both of its nitrogen donors, and a deprotonated oxygen donor at C4 of the sugar moiety. High pressure liquid chromatography (HPLC) of products of 2'-deoxyguanosine (dG) oxidation and agarose gel electrophoresis of plasmid DNA confirmed that lincomycin complexes effectively facilitate dG oxidation by H2O2, but are not able to cleave double-stranded plasmid DNA.     

Copper(II) complexation by pituitary adenylate cyclase activating polypeptide fragments.

Results are reported from potentiometric and spectroscopic (UV-Vis, CD, and ESR) studies of the protonation constants and Cu2+ complex stability constants of pituitary adenylate cyclase activating polypeptide fragments (HSDGI-NH2, TDSYS-NH2, RKQMAVKKYLAAVL-NH2). With HSDGI-NH2, the formation of a dimeric complex Cu2H-2L2 was found in the pH range 5-8, in which the coordination of copper(II) is glycylglycine-like, while the fourth coordination site is occupied by the imidazole N3 nitrogen atom, forming a bridge between two copper(II) ions. The formation of dimeric species does not prevent the deprotonation and coordination of the amide nitrogen, and in pH above 8 the CuH-2L complex is formed. Aspartic acid in the third position of peptide sequence stabilizes the CuH-2L species and prevents the coordination of a fourth nitrogen donor. Aspartic acid residue in the second position of TDSYS-NH2 stabilizes the CuL (2N) complex but does not prevent deprotonation and binding of the second and third peptide nitrogens to give 3N and 4N complexes at higher pH. The tetradecapeptide amide forms with copper(II) ions unusually stable 3N and 4N complexes compared to pentaalanine amide.     

Spectroscopic and potentiometric study of the SOD mimic system copper(II)/acetyl-L-histidylglycyl-L-histidylglycine

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.     

Design of histidine containing peptides for better understanding of their coordination mode toward copper(II) by CD spectroscopy

The systematic investigation of the copper(II) complexes of tripeptides Xaa-Xaa-His, Xaa-His-Xaa and His-Xaa-Xaa, where Xaa=Gly or Ala was performed by combined pH-metry, spectrophotometry, CD and in part EPR spectroscopy. The matrix rank analysis of the spectral data revealed the number of the coloured and optically active species as a basis for the solution speciation. A critical evaluation on the speciation and solution structure of the complexes formed is presented on the basis of their d-d band optical activity. The replacement of a Gly residue with the chiral Ala amino acid allowed us to gain decisive information on the solution structure of the complexes by CD spectroscopy. It was shown that the tripeptides with histidine in the third position formed CuH(-2)L species with (NH(2), 2N(-), ImN - where Im stands for imidazole) coordination sphere as a major species, and only the macrochelated CuL complexes as minor species around pH 5.0. In copper(II)-Xaa-His-Xaa tripeptide systems the CuH(-1)L (NH(2), N(-), ImN) is the most stable species at physiological pH, but the vacant fourth site around copper(II)ions is offered for further deprotonation, most probably resulting in mixed hydroxo species at low (<5 x 10(-4)M) metal ion concentrations, while a tetrameric complex is dominant when the copper concentration exceeds 3 x 10(-3)M. The histamine type coordination mode in CuL and CuL(2) complexes of His-Xaa-Xaa ligands predominates at low pH. The structural consequences drawn from the CD spectra for the mono and bis parent complexes were supported by theoretical calculations. CD spectra strongly suggest the participation of the imidazole nitrogen both in the Cu(2)H(-2)L(2) and CuH(-2)L complexes.     

Nickel(II) binding to Cap43 protein fragments

Cap43 protein has been tested for metal binding domains. The protein, specifically induced by nickel compounds in cultured human cells, had a new mono-histidinic motif consisting of 10 amino acids repeated three times in the C-terminus. The 20-Ac-TRSRSHTSEG-TRSRSHTSEG (Thr(341)-Arg-Ser-Arg-Ser-His(346)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His(356)-Thr-Ser-Glu-Gly(360) - peptide 1) and the 30-Ac-TRSRSHTSEG-TRSRSHTSEG-TRSRSHTSEG (Thr(341)-Arg-Ser-Arg-Ser-His(346)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His(356)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His(366)-Thr-Ser-Glu-Gly(370) - peptide 2) amino acids sequence has been analyzed as a site for Ni(II) binding. A combined pH-metric and spectroscopic (UV-visible, CD, NMR) studies of Ni(II) binding to both fragments were performed. The 20-amino acid peptide can bind one and two metal ions while the 30-amino acid fragment one, two and three metal ions. At physiological pH, depending on the metal to ligand molar ratio, peptide 1 forms the Ni(2)L species while peptide 2 the NiL, Ni(2)L and Ni(3)L complexes where each metal ion is coordinated to the imidazole nitrogen atom of the histidine residue of the 10-amino acid fragment. Octahedral complexes at pH 8-9 and planar 4N complexes with (N(Im), 3N(-)) bonding mode at pH above 9, are formed. This work supports the existence of an interesting binding site at the COOH-terminal domain of the Cap43 protein.     

Solution studies of some new oxamides - co-ordination behaviour towards Cu(II) ions

The co-ordination chemistry of some new oxamides towards Cu(II) ions was studied using various techniques: potentiometry, voltammetry, spectroscopy (UV-Vis, CD and EPR) and ESI-MS spectrometry. All tested compounds chelate the copper(II) ions with formation of 1:1 and 1:2 (metal-to-ligand ratio) complexes. The Cu(II) ions are bound by 1N, 2N or 3N nitrogen donor systems. Additionally, an unusual co-ordination to amide N-atoms without additional anchoring site is suggested. The (14)N hyperfine splitting observed for the system ox6-Cu(II) above pH 10 clearly indicates the involvement of at least three N donor atoms in the copper ion binding. Moreover, the surrounding by three amide-N and one carbonyl-O stabilizes the high oxidation state of copper(III), although such complexes are very unstable in solution.     

Potentiometric and spectroscopic studies on the dimethyltin(IV) complexes of 2-hydroxyhippuric acid

Equilibrium and spectroscopic (1H, 13C NMR and 119Sn M?ssbauer) studies in aqueous solution are reported for dimethyltin(IV) complexes of 2-hydroxyhippuric acid (Sal-Gly). Below pH 4, oxygen-coordinated complexes MLH and ML are formed. In the pH range 5-8.5, the species MLH(-1), predominates at any metal-to-ligand ratio. The ligand exchange of this species is slow on the NMR time scale, which allows its structural characterization by NMR spectroscopy: the coordination polyhedron around the tin atom is distorted trigonal bipyramidal, with tridentate [O-,N-,COO-] coordination of Sal-Gly, involving two equatorial methyl groups. The NMR results reveal that the main cause of the distortion of the polyhedron is the large CH3-Sn-CH3 angle of 136+/-4 degrees. The presented results supplement the data available on the dimethyltin(IV)-promoted amide deprotonation of peptides, and provide further arguments for the fundamental role of the carboxylate as an anchoring group in this process.     

Raman and IR spectroscopic investigation of zinc(II)-carnosine complexes

The zinc(II)-L-carnosine system was investigated at different pH and metal/ligand ratios by Raman and IR spectroscopy. The Raman and IR spectra present some marker bands useful to identify the sites involved in metal chelation at a specific pH value. In particular, the neutral imidazole group gives rise to some Raman bands, such as the nu C(4)===C(5) band, that change in wave number, depending on whether the imidazole ring takes the tautomeric form I or II. Even if tautomer I is predominant in the free ligand, metal coordination can upset tautomeric preference and N(tau)- and N(pi)-ligated complexes can be identified. Although weak compared to those of aromatic residues, these Raman marker bands may be useful in analyzing metal-histidine interaction in peptides and proteins. On the basis of the vibrational results, conclusions can be drawn on the species existing in the system. Depending on the available nitrogen atoms, various complexes can be formed and the prevalent form of the species depends mainly on the pH. At basic pH carnosine gives rise to two different neutral complexes: a water-insoluble polymeric species, [ZnH(-1)L](0)(n), and a dimer, [Zn(2)H(-2)L(2)](0). The first is predominant and involves the tautomeric I form of the imidazole ring in metal chelation; the second contains tautomer II and increases its percentage by going from a 2 to 0.25 metal/ligand ratio. Conversely, the dimeric species dominates at pH 7, whereas two charged species, [ZnHL](2+) and [ZnL](+), are formed under slightly acidic conditions. In the [ZnHL](2+) complex the imidazole ring takes part in the Zn(II) coordination in the tautomeric I form, whereas in [ZnL](+) the ring is protonated and not bound to the Zn(II) ion. In addition, the curve fitting analysis of the 1700-1530 cm(-1) Raman region was helpful in indicating the predominant species at each pH.     

Binding of copper(II) to thyrotropin-releasing hormone (TRH) and its analogs

Spectroscopy (UV-Vis, ¹H NMR, ESR) and electrochemistry revealed details of the structure of the Cu(II)-TRH (pyroglutamyl-histidyl-prolyl amide) complex. The ¹H NMR spectrum of TRH has been assigned. NMR spectra of TRH in the presence of Cu(II) showed that Cu(II) initially binds TRH through the imidazole. TRH analogs, pGlu-His-Pro-OH, pGlu-(1-Me)His-Pro-amide, pGlu-His-(3,4-dehydro)Pro-amide, pGlu-His-OH, pGlu-Glu-Pro-amide, and pGlu-Phe-Pro-amide provided comparison data. The stoichiometry of the major Cu(II)-TRH complex at pH 7.45 and greater is 1:1. The conditional formation constant (in pH 9.84 borate with 12.0 mM tartrate) for the formation of the complex is above 10⁵ M⁻¹. The coordination starts from the 1-N of the histidyl imidazole, and then proceeds along the backbone involving the deprotonated pGlu-His amide and the lactam nitrogen of the pGlu residue. The fourth equatorial donor is an oxygen donor from water. Hydroxide begins to replace the water before the pH reaches 11. Minority species with stoichiometry of Cu-(TRH)_x (x = 2-4) probably exist at pH lower than 8.0. In non-buffered aqueous solutions, TRH acts as a monodentate ligand and forms a Cu(II)-(TRH)₄ complex through imidazole nitrogens. All the His-containing analogs behave like TRH in terms of the above properties.     

Interactions of 1,12-diamino-4,9-dioxadodecane (OSpm) and Cu(II) ions with pyrimidine and purine nucleotides: adenosine-5'-monophosphate (AMP) and cytidine-5'-monophosphate (CMP)

The interactions of Cu(II) ions with adenosine-5'-monophosphate (AMP), cytidine-5'-monophosphate (CMP) and 1,12-diamino-4,9-dioxadodecane (OSpm) were studied. A potentiometric method was applied to determine the composition and stability constants of complexes formed, while the mode of interactions was analysed by spectral methods (ultraviolet and visible spectroscopy (UV-Vis), electron paramagnetic resonance (EPR), (13)C NMR, (31)P NMR). In metal-free systems, molecular complexes nucleotide-polyamine (NMP)H(x)(OSpm) were formed. The endocyclic nitrogen atoms of the purine ring N(1), N(7), the nitrogen atom of the pyrimidine ring N(3), the oxygen atoms of the phosphate group of the nucleotide and the protonated nitrogen atoms of the polyamine were the reaction centres. The mode of interaction of the metal ion with OSpm and the nucleotides (AMP or CMP) in the coordination compounds was established. In the system Cu(II)/OSpm the dinuclear complex Cu(2)(OSpm) forms, while in the ternary systems Cu(II)/nucleotide/OSpm the species type MH(x)LL' and MLL' appear. In the MH(x)LL' type species, the main centres of copper (II) ion binding in the nucleotide are the phosphate groups. The protonated amino groups of OSpm are involved in non-covalent interaction with the nitrogen atoms N(1), N(7) or N(3) of the purine or pyrimidine ring, whereas at higher pH, deprotonated nitrogen atoms of polyamine are engaged in metallation in MLL' species.     

Copper(II) and oxovanadium(IV) complexes of D-3-phosphoglyceric acid

The complexes formed between D-3-phosphoglyceric acid and H(+), Cu(II) and VO(IV) were studied by pH-potentiometric and spectral (UV-Vis, EPR and CD) methods in order to describe the speciation of the metal ions and to determine the most probable binding modes in the complexes formed in these systems. The results show that, in the pH range between 2 and 4, mononuclear 1:1 complexes are formed through bidentate (MAH) or tridentate (MA) coordination of the ligand. At higher pH, when the proton competition for the central alcoholic-OH function decreases, alcoholate-bridged dinuclear species of composition M(2)A(2)H(-n) (n=1-3) become predominant. VO(IV) seems to have a higher tendency than Cu(II) to form such dinuclear complexes.     

Binding of copper(II) to carnosine: Raman and IR spectroscopic study

A comparative Raman and FTIR study of carnosine, a dipeptide present in several mammalian tissues, and its complexes with copper(II) at different pH values was carried out. The neutral imidazole ring gives rise to some bands that appear at different wavenumbers, depending on whether the imidazole ring is in the tautomeric form II or I. At pH 7 and 9 the molecule exists in equilibrium between the two tautomeric forms; tautomer I is predominant. Metal coordination is a factor that affects the tautomeric equilibrium, and the copper(II) coordination site can be monitored by using some Raman marker bands such as the vC(4)=C(5) band. On the basis of the vibrational results, conclusions can be drawn on the functional groups involved in the Cu(II) chelation and on the species existing in the Cu(II)-carnosine system. At neutral and basic pH the most relevant species formed when the Cu(II)/carnosine molar ratio is not very different from unity is a dimer, [Cu(2)L(2)H(-2)](0). In this complex the ligand coordinates the metal via the N (amino), O (carboxylate), and N (amide) donor atoms while the N(tau) nitrogen atoms of the imidazole rings (tautomer II) bridge the copper(II) ions. At a slightly acidic pH the two monomeric complexes [CuLH](2+) and [CuL](+) were present. In the former the imidazole ring takes part in the Cu(II) coordination in the tautomeric I form whereas in the latter it is protonated and not bound to Cu(II).     

Ni(II) and Cu(II) binding with a 14-aminoacid sequence of Cap43 protein, TRSRSHTSEGTRSR

The tetradecapeptide containing the 10 aminoacid repeated sequence on the C-terminus of the Ni(II)-induced Cap43 protein, was analyzed for Ni(II) and Cu(II) binding. A combined pH-metric and spectroscopic UV-VIS, EPR, CD and NMR study of Ni(II) and Cu(II) binding to the blocked CH3CO-Thr-Arg-Ser-Arg-Ser-His-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-NH2 (Ac-TRSRSHTSEGTRSR-Am) peptide, modeling a part of the C-terminal sequence of the Cap43 protein, revealed the formation of octahedral complexes involving imidazole nitrogen of histidine, at pH 5.5 and pH 7 for Cu(II) and Ni(II), respectively; a major square planar 4N-Ni(II) complex (about 100% at pH 9, log K* = -28.16) involving imidazole nitrogen of histidine and three deprotonated amide nitrogens of the backbone of the peptide was revealed; a 3N-Cu(II) complex (maximum about 70% at pH 7, log K*=-13.91) and a series of 4N-Cu(II) complexes starting at pH 5.5 (maximum about 90% at pH 8.7, log K* = -21.39 for CuH(-3)L), were revealed. This work supports the existence of a metal binding site at the COOH-terminal part of the Cap43 peptide.     

Copper(II) complexes with peptide fragments encompassing the sequence 122-130 of human doppel protein

Copper(II) complexes of the peptide fragment (Dpl122-130) encompassing the sequence 122-130 of human doppel protein were characterized by potentiometric, UV-Visible, CD and EPR spectroscopic methods. An analogous peptide, in which the aspartate residue was substituted by an asparagine amino acid, was synthesized in order to provide evidence on the possible role of carboxylate group in copper(II) coordination. It was found that the carboxylic group is directly involved in copper(II) coordination at acidic pH, forming the CuLH₂ species with Dpl122-130. This copper(II) complex displayed EPR parameters very similar to those of the analogous complex with the whole doppel protein. At pH higher than 7, the complexes showed magnetic parameters similar to those of the major species of protein formed in the pH range 7-8, with the metal coordination environment consisting of one imidazole and three amide nitrogen atoms. The comparison of Cu-Dpl122-130 binding constant values with those of the prion peptide fragments (PrP106-114), showed that doppel peptide had a higher metal binding affinity at acidic pH whereas the prion peptide fragment binds the metal tightly at physiological pH.     

The binding of Ni(II) ions to hexahistidine as a model system of the interaction between nickel and His-tagged proteins

The aim of this work is to study the binding of nickel ions to hexahistidine (His(6)) combining potentiometric titrations and spectroscopic (UV-Vis and circular dichroism) determinations in order to establish the species distribution as a function of the pH, their stoichiometry, stability and geometry. For comparative purposes, the same procedure was applied to the Ni-histidine (His) system. His behaves as a tridentate ligand, coordinating the carboxyl group, the imidazole and the amino nitrogen atoms to Ni(II) ions in an octahedral coordination and a bis(histidine) complex is formed at pH higher than 5. For the Ni-His(6) system, the complex formation starts at pH 4 and five different species (Ni(His(6))H, Ni(His(6)), Ni(n)(His(6))(n), Ni(n)(His(6))(n)H(-n/2), Ni(n)(His(6))(n)H(-n)) are formed as a function of the pH. Ni(His(6))H involves the coordination of the imidazole nitrogen and a deprotonated amide nitrogen (N(Im), N(-)) resulting in an octahedral geometry. In Ni(His(6)), an imidazole nitrogen is deprotonated and coordinated (2N(Im), N(-)) to the metal ion with a square planar geometry. The aggregated forms result from the extra Ni-N(Im) coordination, resulting in a 4N square planar geometry that is stabilized by inter/intramolecular hydrogen bonds. This coordination mode is not altered during the deprotonation steps from Ni(n)(His(6))(n).     

Metal-binding ability of histidine-containing peptidehydroxamic acids: Imidazole versus hydroxamate coordination

Three new peptidehydroxamic acids (l-alanyl-l-histidinehydroxamic acid, l-Ala-l-HisNHOH, l-alanyl-l-alanyl-l-histidinehydroxamic acid, l-Ala-l-Ala-l-HisNHOH and l-histidyl-l-alaninehydroxamic acid, l-His-l-AlaNHOH) were synthesized and their complexation with Cu(II), Ni(II) and Zn(II) were studied by pH-potentiometric, UV-Vis, CD, ¹H NMR, EPR and ESI-MS methods. Each of the studied peptide derivatives involves one side-chain imidazole unit and the effect of this group on the metal binding of the hydroxamic moiety is evaluated in the paper. The obtained results are compared to those of the complexes of some histidine-containing di- or tripeptides and also to those of hydroxamic derivatives of aliphatic peptides.A competition between the hydroxamate and imidazole functions occurs in all systems, but the extent differs from metal to metal, from ligand to ligand and depends very much on the pH. The imidazole was found to play the most determinant role in the Cu(II) complexes, somewhat less in the Ni(II)-containing ones, while (except the case of l-Ala-l-HisNHOH) negligible role was found in the Zn(II)-complexes. Common feature of the Ni(II)- and especially Cu(II)-containing systems is that if an imidazole-N is displaced by a hydroxamate, imidazole-bridged di- and polynuclear complexes are formed.     

Solution equilibria and structural characterisation of the palladium(II) and mixed metal complexes of peptides containing methionyl residues

Palladium(II) complexes of the peptides GlyMet, GlyMetGly and GlyGlyMet containing methionyl residues were studied by potentiometric and 1H NMR spectroscopic methods. The coordination of terminal amino and deprotonated amide nitrogen and thioether sulfur donor atoms was suggested in the mono complexes of GlyMet and GlyMetGly. The fourth coordination site of these complexes can be occupied by solvent molecule, chloride or hydroxide ions or by another ligand molecule in the bis or mixed ligand complexes. The second ligand coordinates monodentately via the thioether function in acidic media and the amino group under neutral or basic conditions. The stoichiometry of the major species formed in the palladium(II)-GlyGlyMet system is [PdH(-2) L]- and this is coordinated by the amino, two-amide and the thioether donor functions. Thioether bridged mixed metal complexes formed in the reaction of [Pd(dien)]2+ and [Cu(GlyMetH(-1))] or [Ni(GlyMetGlyH(-2))]- also have been detected by spectroscopic techniques.     

Dipeptides containing the alpha-aminoisobutyric residue (Aib) as ligands: preparation,spectroscopic studies and crystal structures of copper(II) complexes with H-Aib-X-OH (X=Gly,L-Leu,L-Phe)

Synthetic procedures are described that allow access to new copper(II) complexes with dipeptides containing the alpha-aminoisobutyric residue (Aib) as ligands. The solid complexes [Cu(H(-1)L(A))](n).nH(2)O (1) (L(A)H=H-Aib-Gly-OH), [Cu(H(-1)L(B))(MeOH)](n).nMeOH (2) (L(B)H=H-Aib-L-Leu-OH) and [Cu(H(-1)L(C))](n) (3) (L(C)H=H-Aib-L-Phe-OH) have been isolated and characterized by single-crystal X-ray crystallography, solid-state IR spectra and UV-Vis spectroscopy in solution (H(-1)L(2-) is the dianionic form of the corresponding dipeptide). Complexes 1 and 3 are three-dimensional coordination polymers with similar structures. The doubly deprotonated dipeptide behaves as a N(amino), N(peptide), O(carboxylate), O'(carboxylate), O(peptide) mu(3) ligand and binds to one Cu(II) atom at its amino and peptide nitrogens and at one carboxylate oxygen, to a second metal at the other carboxylate oxygen, while a third Cu(II) atom is attached to the peptide oxygen. The geometry around copper(II) is distorted square pyramidal with the peptide oxygen at the apex of the pyramid. The structure of 2 consists of zigzag polymeric chains, where the doubly deprotonated dipeptide behaves as a N(amino), N(peptide), O(carboxylate), O'(carboxylate) mu(2) ligand. The geometry at copper(II) is square pyramidal with the methanol oxygen at the apex. The IR data are discussed in terms of the nature of bonding and known structures. The UV-Vis spectra show that the solid-state structures of 1, 2 and 3 do not persist in H(2)O.     

Synthesis, characterization and crystal structure of copper(II) ternary complex with cinoxacin and histamine

In order to enhance the knowledge of how quinolones may exert their antibacterial action, the synthesis of a new copper(II) mixed ligand, containing histamine complex was carried out. Crystal structure of [Cu(hsm)(cnx)NO₃]H₂O shows that Cu(II) cation displays a distorted square pyramidal geometry. Cinoxacinate (cnx) acts as bidentate ligand bonded to the Cu(II) ion through the oxygen atoms of the 3-carboxylate and 4-keto groups and histamine (hsm) is coordinated by the aliphatic amino group and the non protonated nitrogen of imidazole. Molar conductivity, elemental analysis, infrared and electronic spectroscopy have been used to characterize the complex. The results here reported indicate that cnx coordination mode is similar to that found in other complexes reported previously, being only found that metal-ligand distances are slightly different because the asymmetry of the coordination environment. This result also, may support the hypothesis that the mechanism of action of quinolones could be through a copper(II) complex carrier with imidazole ligands in their coordination environment.     

Acid-base properties and copper(II) complexes of dipeptides containing histidine and additional chelating bis(imidazol-2-yl) residues

Copper(II) complexes of dipeptides of histidine containing additional chelating bis(imidazol-2-yl) agent at the C-termini (PheHis-BIMA [N-phenylalanyl-histidyl-bis(imidazol-2-yl)methylamine] and HisPhe-BIMA [N-histidyl-phenylalanyl-bis(imidazol-2-yl)methylamine]) were studied by potentiometric, UV-Visible and Electron Paramagnetic Resonance (EPR) techniques. The imidazole nitrogen donor atoms of the bis(imidazol-2-yl)methyl group are described as the primary metal binding sites forming stable mono- and bis(ligand) complexes at acidic pH. The formation of a ligand-bridged dinuclear complex [Cu2L2]4+ is detected in equimolar solutions of copper(II) and HisPhe-BIMA. The coordination isomers of the dinuclear complex are described via the metal binding of the bis(imidazol-2-yl)methyl, amino-carbonyl and amino-imidazole(His) functions. In the case of the copper(II)-PheHis-BIMA system the [NH2, N-(amide), N(Im)] tridentate coordination of the ligand is favoured and results in the formation of di- and trinuclear complexes [Cu2H(-1)L]3+ and [Cu3H(-2)L2]4+ in equimolar solutions. The presence of these coordination modes shifts the formation of "tripeptide-like" ([NH2, N-, N-, N(Im)]-coordinated) [CuH(-2)L] complexes into alkaline pH range as compared to other dipeptide derivatives of bis(imidazol-2-yl) ligands. Although there are different types of imidazoles in these ligands, the deprotonation and coordination of the pyrrole-type N(1)H groups does not occur below pH 10.