Impact of ring size on the copper(II) coordination abilities of cyclic tetrapeptides

A new, 14-membered, tetraza cyclic tetrapeptide containing histidine and lysine side-chains, c(β³homoLysdHisβ-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.     

Modeling of copper(II) sites in proteins based on histidyl and glycyl residues

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.     

The copper(II) binding properties of the cyclic peptide c(HGHK)

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 cyclopeptides with the multi-His motif as ligands for copper(II)

The coordination properties of cyclic octapeptides with multi-His motif: c(His-Gly-His-Xaa-His-Gly-His-Xaa) where Xaa = Asp or Lys, were investigated. The binding abilities of this peptides towards Cu(II) ions were studied by using different analytic methods as: potentiometry, spectroscopy and mass spectrometry. The obtained results show that the studied peptides in physiological related pH prefer formation of the species with the {4N_Im} binding mode. The efficiency of Cu(II) binding depends on additional side chain groups Asp or Lys. Additionally the analysis of results for His containing cyclopeptides with different numbers of amino acid residues in cyclopeptide ring e.g. four, eight shows that in higher pH in both cases the binding by four amide nitrogens is not observed in the case of α-amino acid peptides.     

Short-chain oligopeptides with copper(II) binding properties: The impact of specific structural modifications on the copper(II) coordination abilities

A series of linear tetrapeptides containing two histidyl residues in position 2 and 4, namely DHGH, DHGdH, KHGH, KHGdH, Ac-DHGH-NH₂, Ac-DHGdH-NH₂, Ac-KHGH-NH₂, and Ac-KHGdH-NH₂, 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.     

The effect of histidyl residues on the complexation of bis(imidazolyl) containing tripeptides with copper(II) ion

Copper(II) complexes of tripeptide derivatives of bis(imidazol-2-yl) group have been studied by potentiometric, UV-visible and EPR spectroscopic methods. The peptide molecules correspond to the amino acid sequence of collagen containing histidyl residues in different locations and were connected to the bis(imidazol-2-yl) group either on the C-termini (BOC-Pro-Leu-His-BIMA, BOC-His-Leu-Gly-BIMA) or on the N-termini (BIP-His-Ala-Gly-OEt, BIP-Ile-Ala-His-OMe). It was concluded that the imidazole nitrogen donor atoms of the bis(imidazol-2-yl) moiety are the primary metal binding sites, but the histidyl imidazole nitrogens in the side chains have also some effect on the stability and the coordination mode of the complexes. All ligands can coordinate tridentately to copper(II) ion forming a six-membered chelate and a macrochelate in the [CuL]2+ complexes, which results in a slight distortion in the coordination geometry of [CuL2]2+ complexes. The deprotonation and coordination of amide nitrogens, however, were not observed in any cases.     

Cu(II)–protamine interaction. II. The formation and structure of Cu(II) complexes of clupeine YII and of peptides mimicking clupeine N-terminals

The interaction of Cu(II) with the protamine clupeine YII (containing proline at the N-terminal) and with four peptides (H-Ala-Arg-OMe, H-Ala-Arg₂-OMe, H-Pro-Arg-OMe, and H-Arg₄-Tyr) has been studied by means of absorption, CD, and pH neasurements. The first two peptides mimic clupeine YI and Z N-terminals; the third, the clupeine YII N-terminal. At 1:1 molar ratio, clupeine YII yields two complexes: the first (I), at pH 6.6, through coordination via the N-terminal and the contiguous peptide nitrogen forming a five-membered chelate; the second (II), at pH 8.5, through the occupancy of the other two corners of the coordination square by amino nitrogens of the lateral chains. These complexes are strictly analogous and occur at the same pH as those formed with clupeine Z. Under the same conditions, all the peptides yield complex I in the first step, although the pH at which this complex is fully defined depends on the number of residues in the chain. It is 8.5 for dipeptides, decreases to 6.5 by the addition of a third residue to the chain, and remains constant when the number of residues is three or more. The amino nitrogens of lateral chains are unable to coordinate to the metal in a second step unless one additional peptide bond lies between the N-terminal residue and that containing the lateral chain bound to the metal. Thus, H-Ala-Arg-OMe and H-Pro-Arg-OMe form hydroxyl complexes in a second step (pH 11), by deprotonation of one of the water molecules coordinated to the metal; one of the lateral chains of H-Ala-Arg₂-OMe is able to coordinate in a second step (pH 8.5), but it is only with H-Arg₄-Tyr that a second complex (II) is obtained in which two amino nitrogens of lateral chains supersede the oxygens of water molecules in I, at pH 8.5.     

Copper(II) complexes of 1,10-phenanthroline-derived ligands: studies on DNA binding properties and nuclease activity

A series of copper(II) complexes of the type [Cu(L)]2+, where L = N,N'-dialkyl-1,10-phenanthroline-2,9-dimethanamine and R = methyl (L1), n-propyl (L2), isopropyl (L3), sec-butyl (L4), or tert-butyl (L5) group, have been synthesized. The interaction of the complexes with DNA has been studied by DNA fiber electron paramagnetic resonance (EPR) spectroscopy, emission, viscosity and electrochemical measurements and agarose gel electrophoresis. In the X-ray crystal structure of [Cu(HL2)Cl2]NO3, copper(II) is coordinated to two ring nitrogens and one of the two secondary amine nitrogens of the side chains and two chloride ions as well and the coordination geometry is best described as trigonal bipyramidal distorted square based pyramidal (TBDSBP). Electronic and EPR spectral studies reveal that all the complexes in aqueous solution around pH 7 possess CuN3O2 rather than CuN4O chromophore with one of the alkylamino side chain not involved in coordination. The structures of the complexes in aqueous solution around pH 7 change from distorted tetragonal to trigonal bipyramidal as the size of the alkyl group is increased. The observed changes in the physicochemical features of the complexes on binding to DNA suggest that the complexes, except [Cu(L5)]2+, bind to DNA with partial intercalation of the derivatised phen ring in between the DNA base pairs. Electrochemical studies reveal that the complexes prefer to bind to DNA in Cu(II) rather than Cu(I) oxidation state. Interestingly, [Cu(L5)]2+ shows the highest DNA cleavage activity among all the present copper(II) complexes suggesting that the bulky N-tert-butyl group plays an important role in modifying the coordination environment around the copper(II) center, the Cu(II)/Cu(I) redox potential and hence the formation of activated oxidant responsible for the cleavage. These results were compared with those for bis(1,10-phenanthroline)copper(II), [Cu(phen)2]2+.     

Nickel(II) complexes of the multihistidine peptide fragments of human prion protein

Nickel(II) complexes of the peptide fragments of human prion protein containing histidyl residues both inside and outside the octarepeat domain have been studied by the combined application of potentiometric, UV-visible and circular dichroism spectroscopic methods. The imidazole-N donor atoms of histidyl residues are the exclusive metal binding sites below pH 7.5, but the formation of stable macrochelates was characteristic only for the peptide HuPrP(76-114) containing four histidyl residues. Yellow colored square planar complexes were obtained above pH 7.5-8 with the cooperative deprotonation of three amide nitrogens in the [N_im,N⁻,N⁻,N⁻] coordination mode. It was found that the peptides can bind as many nickel(II) ions as the number of independent histidyl residues. All data supported that the complex formation processes of nickel(II) are very similar to those of copper(II), but with a significantly reduced stability for nickel(II), which shifts the complex formation reactions into the slightly alkaline pH range. The formation of coordination isomers was characteristic of the mononuclear complexes with a significant preference for the nickel(II) binding at the histidyl sites outside the octarepeat domain. The results obtained for the two-histidine fragments of the protein, HuPrP(91-115), HuPrP(76-114)H85A and HuPrP(84-114)H96A, made it possible to compare the binding ability of the His96 and His111 sites. These data reveal a significant difference in the nickel(II) and copper(II) binding sites of the peptides: His96 was found to predominate almost completely for nickel(II) ions, while the opposite order, but with comparable concentrations, was reported for copper(II).     

Anticancer potency of small linear and cyclic tetrapeptides and pharmacokinetic investigations of peptide binding to human serum albumin

We have in the present study explored the anticancer activity against human Burkitt's lymphoma cells (Ramos) of a series of small linear and cyclic tetrapeptides containing a β^2,2‐amino acid with either two 2‐naphthyl‐methylene or two para‐CF₃‐benzyl side chains, along with their interaction with the main plasma protein human serum albumin (HSA). The cyclic and more amphipathic tetrapeptides revealed a notably higher anticancer potency against Ramos cells [50% inhibitory concentration (IC₅₀) 11–70 μM] compared to the linear tetrapeptide counterparts (IC₅₀ 18.7 to >413 μM). The most potent cyclic tetrapeptide c3 had a 16.5‐fold preference for Ramos cells compared to human red blood cells, whereas the cyclic tetrapeptide c1 both showed low hemolytic activity and displayed the overall highest (2.9‐fold) preference for Ramos cells (IC₅₀ 23 μM) compared to healthy human lung fibroblast cells (MRC‐5). Investigating the interaction of selected tetrapeptides and recently reported hexapeptides with HSA revealed that the peptides bind to drug site II of HSA in the 22–28 μM range, disregarding size and overall structure. NMR and in silico molecular docking experiments identified the lipophilic residues as responsible for the interaction, but in vitro studies showed that the anticancer potency of the peptides varied in the presence of HSA and that c3 remained the most potent peptide. Based on our findings, we call for implementing serum albumin binding in development of anticancer peptides, as it may have implications for future administration and systemic distribution of peptide‐based cancer drugs. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Copper-glutathione complexes under physiological conditions: structures in solution different from the solid state coordination

The physiologically important copper complexes of oxidized glutathione have been examined by electron spin resonance (ESR) spectroscopy in aqueous solution at neutral pH. Low temperature measurements show that the Cu(II) binding site in oxidized glutathione has the same ligand arrangement as in the copper complexes of S-methylglutathione, glutamine, glutamate and glycine. The site is composed of the amino nitrogens and the carboxyl oxygens of two -glutamyl residues; there is no interaction with amide nitrogens, the sulphur bond or the glycyl carboxyl groups. At high metal to ligand ratios a binuclear species exists, in which each Cu(II) binds only to one -glutamyl residue. The previously reported forbidden transition detected at g = 4 is due to non-specific aggregation and not to spin coupling of intramolecular sites. Liquid solution ESR spectra show the Cu(II)-glutathione complex has a lower mobility than the corresponding Cu(II)-S'-methylglutathione species. From the degree of spectral anisotropy the complex with glutathione is calculated to exist as a dimer. These results demonstrate that the physiologically relevant complex between copper and oxidized glutathione in solution is completely different from the known solid state structure determined by crystallography.     

Isolation and two-dimensional 1H-NMR of peptide [1-24] of dog serum albumin and studies of its complexation with copper and nickel by NMR and CD spectroscopy

The NH2-terminal peptide fragment [1-24] of dog serum albumin was obtained by controlled peptic digestion of the protein. The peptide was purified to homogeneity by gel filtration and ion-exchange chromatography. The NMR assignments of the protons of the individual amino acid residues were made by using two-dimensional correlation matrix, spin-decoupling experiments and analysis of the titration curves. The polypeptide itself has a random-coil conformation. There is a conformational change as a function of pH, but it does not arise from any direct involvement of the amino acid side chains. Complexation of the peptide fragment with Ni(II) and Cu(II) has been investigated by NMR and CD. The Ni(II) complex is in slow exchange with the free ligand on the NMR time scale. The complexation involves the alpha-NH2, three deprotonated amide nitrogens of Ala-2, Tyr-3 and Lys-4 residues. The phenolate oxygen of Tyr-3 is not involved in the metal binding; however, an interaction between the aromatic ring and the metal ion is likely. The CD results of Cu(II)-binding to this peptide suggest that the complexation takes place from the terminal NH2 and step by step to three deprotonated amide nitrogens. There is no major conformational change of the peptide fragment upon complexation.     

Speciation study of an imidazolate-bridged copper(II)-zinc(II) complex in aqueous solution

The solution equilibrium and the binding mode of the species in the five-component system containing two metal ions (copper(II) and zinc(II)) and three ligands (A=diethylenetriamine, B=imidazole, C=tris(2-aminoethyl)amine) were investigated by pH-potentiometric titration, UV-visible spectrophotometry and EPR (electron paramagnetic resonance) spectroscopic titration in aqueous solution in the 2-11 pH range. An imidazolate-bridged heterobinuclear complex (ACuBH(-1)ZnC) was found to evolve above pH=7 and was stable between pH 7 and 11. The existence of the ACuBH(-1)ZnC complex (by determination of its molecular weight) was proved by mass spectrometry (ESI-MS (electrospray ionization mass spectrometry) and MALDI (matrix-assisted laser desorption/ionization) techniques). The electrochemical behaviour and the superoxide dismutase activity of this complex were also tested by cyclic voltammetry and the Riboflavin/NBT (nitro blue tetrazolium) assay, respectively.     

Catechol Oxidase and SOD Mimicking by Copper(II) Complexes of Multihistidine Peptides

Copper(II) complexes of five peptide ligands containing at least three histidine residues have been tested as catalysts in catechol oxidation and superoxide dismutation. All systems exhibit considerable catechol oxidase-like activity, and the Michaelis–Menten enzyme kinetic model is applicable in all cases. Beside the Michaelis–Menten parameters, the effects of pH, catalyst and dioxygen concentration on the reaction rates are also reported. Considering the rather different sequences, the observed oxidase activity seems to be a general behavior of copper(II) complexes with multihistidine peptides. Interestingly, in all cases {N_im/2N_im,2N^?} coordinated complexes are the pre-active species, the bound amide nitrogens were proposed to be an acid/base site for facilitating substrate binding. The studied copper(II)-peptide complexes are also able to effectively dismutate superoxide radical in the neutral pH range.     

Tandem repeat-like domain of "similar to prion protein" (StPrP) of Japanese pufferfish binds Cu(II) as effectively as the mammalian protein

The main structural domains of prion proteins, in particular the N-terminal region containing characteristic amino acid repeats, are well conserved among different species, despite divergence in primary sequence. The repeat region seems to play an important role, as verified by pathogenicity only observed in organisms having repeats composed of eight residues. In this work three different peptides belonging to the tandem repeat region of StPrP-2 from the Japanese pufferfish Takifugu rubripes have been considered; the coordination modes and conformations of their complexes with Cu(II) have been investigated by using potentiometric titrations, spectroscopic data, and restrained molecular dynamics simulations. In all cases the histidine imidazole(s) provide the anchoring site for copper, with the further involvement of amide nitrogens depending on the peptide sequence and on pH. An increase in copper binding affinity has been observed going from the shortest peptide, corresponding to a single repeat and containing two histidines, to the longest one, encompassing three repeats with six histidines.     

Raman spectroscopic study on the copper(II) binding mode of prion octapeptide and its pH dependence.

The cellular form of prion protein is a precursor of the infectious isoform, which causes fatal neurodegenerative diseases through intermolecular association. One of the characteristics of the prion protein is a high affinity for Cu(II) ions. The site of Cu(II) binding is considered to be the N-terminal region, where the octapeptide sequence PHGGGWGQ repeats 4 times in tandem. We have examined the Cu(II) binding mode of the octapeptide motif and its pH dependence by Raman and absorption spectroscopy. At neutral and basic pH, the single octapeptide PHGGGWGQ forms a 1:1 complex with Cu(II) by coordinating via the imidazole N pi atom of histidine together with two deprotonated main-chain amide nitrogens in the triglycine segment. A similar 1:1 complex is formed by each octapeptide unit in (PHGGGWGQ)2 and (PHGGGWGQ)4. Under weakly acidic conditions (pH approximately 6), however, the Cu(II)-amide- linkages are broken and the metal binding site of histidine switches from N pi to N tau to share a Cu(II) ion between two histidine residues of different peptide chains. The drastic change of the Cu(II) binding mode on going from neutral to weakly acidic conditions suggests that the micro-environmental pH in the brain cell regulates the Cu(II) affinity of the prion protein, which is supposed to undergo pH changes in the pathway from the cell surface to endosomes. The intermolecular His(N tau)-Cu(II)-His(N tau) bridge may be related to the aggregation of prion protein in the pathogenic form.     

Identification of the Cu2+ binding sites in the N-terminal domain of the prion protein by EPR and CD spectroscopy

Recent evidence indicates that the prion protein (PrP) plays a role in copper metabolism in the central nervous system. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60-91. This region selectively binds divalent copper ions (Cu(2+)) in vivo. To elucidate the specific mode and site of binding, we have studied a series of Cu(2+)-peptide complexes composed of 1-, 2-, and 4-octarepeats and several sub-octarepeat peptides, by electron paramagnetic resonance (EPR, conventional X-band and low-frequency S-band) and circular dichroism (CD) spectroscopy. At pH 7.45, two EPR active binding modes are observed where the dominant mode appears to involve coordination of three nitrogens and one oxygen to the copper ion, while in the minor mode two nitrogens and two oxygens coordinate. ESEEM spectra demonstrate that the histidine imidazole contributes one of these nitrogens. The truncated sequence HGGGW gives EPR and CD that are indistinguishable from the dominant binding mode observed for the multi-octarepeat sequences and may therefore comprise the fundamental Cu(2+) binding unit. Both EPR and CD titration experiments demonstrate rigorously a 1:1 Cu(2+)/octarepeat binding stoichiometry regardless of the number of octarepeats in a given peptide sequence. Detailed spin integration of the EPR signals demonstrates that all of the bound Cu(2+) is detected thereby ruling out strong exchange coupling that is often found when there is imidazolate bridging between paramagnetic metal centers. A model consistent with these data is proposed in which Cu(2+) is bound to the nitrogen of the histidine imidazole side chain and to two nitrogens from sequential glycine backbone amides.     

Copper(II), nickel(II) and zinc(II) complexes of N-acetyl-His-Pro-His-His-NH2: equilibria, solution structure and enzyme mimicking

The copper(II), nickel(II) and zinc(II) binding ability of the multi-histidine peptide N-acetyl-His-Pro-His-His-NH₂ has been studied by combined pH-potentiometry and visible, CD and EPR spectroscopies. The internal proline residue, preventing the metal ion induced successive amide deprotonations, resulted in the shift of this process toward higher pH values as compared to other peptides. The metal ions in the parent [ML]²⁺ complexes are exclusively bound by the three imidazole side chains. In [CuH₋₁L]⁺, formed between pH 6-8, the side chains of the two adjacent histidines and the peptide nitrogen between them are involved in metal ion binding. The next deprotonation results in the proton loss of the coordinated water molecule (CuH₋₁L(OH)). The latter two species exert polyfunctional catalytic activity, since they possess superoxide dismutase-, catecholase- (the oxidation of 3,5-di-tert-butylcatechol) and phosphatase-like (transesterification of the activated phosphoester 2-hydroxypropyl-4-nitrophenyl phosphate) properties. On further increase of the pH rearrangement of the coordination sphere takes place leading to the [CuH₋₃L]⁻ species, the deprotonated amide nitrogen displaces a coordinated imidazole nitrogen from the equatorial position of the metal ion. The shapes of the visible and CD spectra reflect a distorted arrangement of the donor atoms around the metal ion. In presence of zinc(II) the species [ZnL]²⁺ forms only above pH 6, which is shortly followed by precipitation. On the other hand, the [NiL]²⁺ complex is stable over a wide pH range, its deprotonation takes place only above pH 8. At pH 10 an octahedral NiH₋₂L species is present at first, which transforms slowly to a yellow square planar complex.     

Cu(II)–Protamine interaction. I. The formation and structure of Cu(II)–clupeine Z complexes

The formation and structure of four different Cu(II)–clupeine Z complexes have been studied using potentiometric and spectroscopic (ir and visible absorption, and CD) measurements. The results thus obtained indicate the presence of up to 8 binding sites in the pH range from 6.5 to 10.5. The spectroscopic evidence suggests that the strongest site available contains the α-amino terminal and the adjacent peptide nitrogen, which bind to copper from pH 5 to 6.5 to form the first complex. The stability constant of this first complex has a value of (9.5 ± 0.9) × 10³ mol^?1 1. From pH 6.5 to 8.5, two intervening guanidinium nitrogens of arginine residues occupy the two other corners of the coordination square, giving rise to the second complex. The other sites potentially available from pH 6.5 to 10.5 are formed by two amino nitrogens of arginine residues and two contiguous peptide nitrogens. The first intervene up to pH 8.5, forming the third complex, and the latter from this pH to 10.5, forming the fourth complex. Although the ligands intervening at sites 2–8 appear to be the same, the sites are by no means equivalent. The spectroscopic data enable one to distinguish three different types of binding sites.     

Synthesis and properties of different metal complexes of the siderophore desferriferricrocin

Desferriferricrocin is a cyclic hexa-peptide siderophore with three hydroxamates as primary coordination groups. It forms metal complexes with Fe(III), Cr(III), Al(III), Ga(III), Cu(II), and Zn(II). These complexes were prepared and characterized using UV–vis, circular dichroism spectroscopy (CD), nuclear magnetic resonance spectroscopy (NMR), and electrospray ionization mass spectroscopy (ESI-MS). The mononuclear trivalent metal complexes of desferriferricrocin were stable in aqueous solutions, and their coordination centers primarily adopted the Λ configuration. The formation of multinuclear complexes of desferriferricrocin was determined by ESI-MS. Desferriferricrocin was able to bind up to three Cu(II) and two Zn(II) respectively. Heteronuclear complexes containing one trivalent and one divalent were also determined. In these complexes, amide nitrogens were utilized as alternative binding groups of desferriferricrocin in addition to the primary binding groups, the hydroxamates. Published online December 2004