Novel trihydroxamate-containing peptides: design, synthesis, and metal coordination

Novel trihydroxamate-containing peptides were designed to mimic desferrioxamine (Desferal(R), DFO, a naturally occurring siderophore) but possess distinct conformational restrictions and varied lipophilicity to probe structure vs. metal coordination. The synthesis was performed via fragment condensation of hydroxamate-containing oligopeptides such as Fmoc-Leu- Psi[CON(OBz)]-Phe-Ala-Pro-OH and H-Leu-Psi[CON(OBz)]-Phe-Ala-Pro-OBu(t) (Fmoc: 9-fluor enylmethoxycarbonyl; OBz: benzyl; OBu(t): tert-butyl) either in solution or on a solid support. The metal-binding properties were studied by electrospray ionization-mass spectroscopy (ESI-MS), ultraviolet (UV)-visible spectroscopy, and (1)H nuclear magnetic resonance (NMR). Similar to the dihydroxamate analogs previously explored [Biopolymers (Peptide Science), 2003, Vol. 71, pp. 489-515], the compounds with three hydroxamates arrayed at 10-atom intervals, i.e., H-[Leu-Psi[CON(OH)]-Phe-Ala-Pro](3)-OH (P1), cyclo[Leu-Psi[CON(OH)]-Phe-Ala-Pro](3) (P2), and H-[Leu-Psi(CONOH)-Phe-Ala-Pro](2)-Leu-NHOH (P7), exhibited high affinities for intramolecular coordination with Fe(III) and Ga(III). As expected, both P1 and P2 showed higher relative Fe(III)-binding affinities than the corresponding dihydroxamate-containing peptide analogs (P11 and P12). Even though both P1 and P2 did not compete with DFO in the relative metal-binding affinity in both solution and gas phases, P1, P2, and DFO exhibited similar relative binding selectivities to 11 different metal ions including Fe(III), Fe(II), Al(III), Ga(III), In(III), Zn(II), Cu(II), Co(II), Ni(II), Gd(III), and Mn(II). Compared to the other metal ions, they had higher relative binding affinities with Fe(III), Fe(II), Al(III), Ga(III), and In(III). The decreased metal-binding affinities of P1 and P2 in comparison with DFO suggested the conformational restrictions of their backbones perturb their three hydroxamate groups from optimal hexadentate orientations for metal coordination. As detected by ESI-MS, P2 was distinguished from both P1 and DFO by solvation of its Ga(III) and Fe(III) complexes (such as acetonitrile or water), thereby stabilizing the resulting complexes in the gas phase. Noteworthy, P2 led to 69% death rate in Hela cells at a concentration of 50 microM, exhibiting higher cytotoxicity than DFO in vitro despite its much lower affinity for iron. This enhanced toxicity may simply reflect the increased lipophilicity of the cyclic trihydroxamate (P2) together with the improvements in its cell penetration, and/or subsequent intracellular molecular recognition of both side chains and hydroxamate groups. The cytotoxicity was significantly suppressed by precoordination with Ga(III) or Fe(III), suggesting a mechanism of toxicity via sequestration of essential metal ions as well as the importance of curbing the metal coordination before targeting. The potential of such siderophore-mimicking peptides in oncology needs further exploration.     

Complexation of desferricoprogen with trivalent Fe, Al, Ga, In and divalent Fe, Ni, Cu, Zn metal ions: effects of the linking chain structure on the metal binding ability of hydroxamate based siderophores

Complexes of the natural siderophore, desferricoprogen (DFC), with several trivalent and divalent metal ions in aqueous solution were studied by pH-potentiometry, UV-Vis spectrophotometry and cyclic voltammetry. DFC was found to be an effective metal binding ligand, which, in addition to Fe(III), forms complexes of high stability with Ga(III), Al(III), In(III), Cu(II), Ni(II) and Zn(II). Fe(II), however, is oxidized by DFC under anaerobic conditions and Fe(III) complexes are formed. By comparing the results with those of desferrioxamine B (DFB), it can be concluded that the conjugated beta-double bond slightly increases the stability of the hydroxamate chelates, consequently increases the stability of mono-chelated complexes of DFC. Any steric effect by the connecting chains arises only in the bis- and tris-chelated complexes. With metal ions possessing a relatively big ionic radius (Cu(II), Ni(II), Zn(II), In(III)) DFC, containing a bit longer chains than DFB, forms slightly more stable complexes. With smaller metal ions the trend is the opposite. Also a notable difference is that stable trinuclear complex, [Cu(3)L(2)], is formed with DFC but not with DFB. Possible bio-relevance of the Fe(II)/Fe(III) results is also discussed in the paper.     

Demonstration of nitrogen coordination in metal--bleomycin complexes by electron spin--echo envelope spectroscopy

We have studied the Cu(II), Co(II), and Fe(III) complexes of the antineoplastic drug bleomycin by using electron spin--echo envelope spectroscopy. For all three complexes, nitrogen coordination of the metal ions is demonstrated. For the Cu(II)-- and Co(II)--drug complexes, we have been able to identify imidazole as a metal ligand.     

Zinc(II) modulates specifically amyloid formation and structure in model peptides

Metal ions such as zinc and copper can have dramatic effects on the aggregation kinetics of and the structures formed by several amyloidogenic peptides/proteins. Depending on the identity of the amyloidogenic peptide/protein and the conditions, Zn(II) and Cu(II) can promote or inhibit fibril formation, and in some cases these metal ions have opposite effects. To better understand this modulation of peptide aggregation by metal ions, the impact of Zn(II) binding to three amyloidogenic peptides (Aβ14-23, Aβ11-23, and Aβ11-28) on the formation and structure of amyloid-type fibrils was investigated. Zn(II) was able to accelerate fibril formation for all three peptides as measured by thioflavin T fluorescence and transmission electron microscopy. The effects of Zn(II) on Aβ11-23 and Aβ11-28 aggregation were very different compared with the effects of Cu(II), showing that these promoting effects were metal-specific. X-ray absorption spectroscopy suggested that the Zn(II) binding to Aβ11-23 and Aβ11-28 is very different from Cu(II) binding, but that the binding is similar in the case of Aβ14-23. A model is proposed in which the different coordination chemistry of Zn(II) compared with Cu(II) explains the metal-specific effect on aggregation and the difference between peptides Aβ14-23 and Aβ11-23/Aβ11-28.     

Metal binding modes of Alzheimer's amyloid beta-peptide in insoluble aggregates and soluble complexes

Aggregation of the amyloid beta-peptide (Abeta) into insoluble fibrils is a key pathological event in Alzheimer's disease. Zn(II) induces the Abeta aggregation at acidic-to-neutral pH, while Cu(II) is an effective inducer only at mildly acidic pH. We have examined Zn(II) and Cu(II) binding modes of Abeta and their pH dependence by Raman spectroscopy. The Raman spectra clearly demonstrate that three histidine residues in the N-terminal hydrophilic region provide primary metal binding sites and the solubility of the metal-Abeta complex is correlated with the metal binding mode. Zn(II) binds to the N(tau) atom of the histidine imidazole ring and the peptide aggregates through intermolecular His(N(tau))-Zn(II)-His(N(tau)) bridges. The N(tau)-metal ligation also occurs in Cu(II)-induced Abeta aggregation at mildly acidic pH. At neutral pH, however, Cu(II) binds to N(pi), the other nitrogen of the histidine imidazole ring, and to deprotonated amide nitrogens of the peptide main chain. The chelation of Cu(II) by histidine and main-chain amide groups results in soluble Cu(II)-Abeta complexes. Under normal physiological conditions, Cu(II) is expected to protect Abeta against Zn(II)-induced aggregation by competing with Zn(II) for histidine residues of Abeta.     

Biosorption of heavy metals by the exopolysaccharide produced by <Emphasis Type="Italic">Paenibacillus jamilae</Emphasis>

The biosorption of several toxic heavy metals (Pb, Cd, Co, Ni, Zn and Cu) by the exopolysaccharide (EPS) produced by Paenibacillus jamilae, a potential biosorbent for metal remediation and recovery was studied. Firstly, the biochemical composition of this bacterial polymer was determined. Glucose was the most abundant neutral sugar, followed by galactose, rhamnose, fucose and mannose. The polymer presented a high content of uronic acids (28.29%), which may serve as binding sites for divalent cations. The presence of carboxylic groups was also detected by infrared spectroscopy. The EPS presented an interesting affinity for Pb in comparison with the other five metals. Lead biosorption (303.03 mg g⁻¹) was tenfold higher (in terms of mg of metal adsorbed per gram of EPS) than the biosorption of the rest of metals. Biosorption kinetics, the effect of pH and the effect of competitive biosorption were determined. Finally, we found that the EPS was able to precipitate Fe(III), but the EPS-metal precipitate did not form with Fe(II), Pb(II), Cd(II), Co(II), Ni(II), Cu(II) and Zn(II).     

Unfolding of iron and copper complexes of human lactoferrin and transferrin

1. Human lactoferrin and transferrin are capable of binding two iron or copper ions into specific binding sites in the presence of bicarbonate. 2. Urea and several alkyl ureas have been effective in unfolding these metal-protein complexes. 3.Biphasic transitions are observed for the unfolding of each of the metal complexes of these proteins as determined by direct visible spectroscopy suggesting the release of iron(III) and Cu(II) ions from both of these metal-binding proteins during the unfolding process. 4. Greater stabilization and increased resistance to protein unfolding is observed for all iron(III) complexes compared to Cu(II) complexes of lactoferrin and transferrin as determined by isothermal unfolding and thermal denaturation. 5. Relative stabilization of the different metal-protein complexes investigated within this study were determined to be as follows: Lf-Fe(III) greater than Lf-Cu(II); Tf-Fe(III) greater than Tf-Cu(II), and Lf-Fe(III) greater than Tf-Fe(III); Lf-Cu(II) greater than Tf-Cu(II).     

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.     

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.     

Probing copper/tau protein interactions electrochemically

Alzheimer’s disease (AD) is a neurodegenerative disorder that is characterized by peptide and protein misfolding and aggregation, in part due to the presence of excess metal ions such as copper(II) [Cu(II)]. Recently, the brain levels of Cu(II) complexes in vivo were linked to the oxidative stress in neurodegenerative disorders, including AD. Amyloid β-peptide (Aβ), found outside neuronal cells, has been investigated extensively in connection with Cu(II) ion toxicity; however, the effects of metallation on tau are less known. Normal tau protein binds and stabilizes the microtubules in neurons, but in diseased cells tau hyperphosphorylation and aggregation are evident and compromise tau function. There is increasing evidence that the Cu(II) ion may play an important role in tau biochemistry. Here, we present an electrochemical study of the interactions between full-length tau-410 and Cu(II) ions. The coordination of Cu(II) ions to tau immobilized on gold surfaces induces an electrochemical signal at approximately 140 ± 5 mV versus Ag/AgCl due to the Cu(II)/Cu(I) redox couple. Redox potentials and current intensities of Cu(II)-containing nonphosphorylated tau (nTau) and phosphorylated tau (pTau) films were determined at different pH conditions. Greater Cu(II) uptake by pTau over nTau films was observed at low pH. Competitive zinc(II) [Zn(II)] ion binding studies revealed significant Cu(II) ion displacement in pTau films. X-ray photoelectron spectroscopy analysis indicated the presence of Cu 2p and Zn 2p binding energies in protein samples, further supporting metal ion coordination to protein films. The surface-based electrochemical technique requires a minimal protein amount (a few microliters) and allows monitoring the bound Cu(II) ions and the redox activities of the resulting metalloprotein films.     

Synthesis and characterization of a series of new mixed ligand complexes of manganese(III), iron(III), nickel(II), copper(II) and zinc(II) with schiff bases of N,N-diethylamino-dithiocarbamate as ligands

Twenty new bioactive complexes of Mn(III), Fe(III), Ni(II), Cu(II) and Zn(II) have been prepared containing Schiff bases of N,N-diethylaminodithio- carbamate as ligands. These complexes have been characterized by elemental analyses, IR and UV-Vis spectroscopy as well as by magnetic susceptibility measurements. The spectra of the complexes suggest that the ligands are coordinated to the metal ions via the sulfur atoms of the dithiocarbamato group.     

Identifying the minimal copper- and zinc-binding site sequence in amyloid-beta peptides

With a combination of complementary experimental techniques, namely sedimentation assay, Fourier transform infrared spectroscopy, and x-ray absorption spectroscopy, we are able to determine the atomic structure around the metal-binding site in samples where amyloid-beta (Abeta) peptides are complexed with either Cu(II) or Zn(II). Exploiting information obtained on a selected set of fragments of the Abeta peptide, we identify along the sequence the histidine residues coordinated to the metal in the various peptides we have studied (Abeta(1-40), Abeta(1-16), Abeta(1-28), Abeta(5-23), and Abeta(17-40)). Our data can be consistently interpreted assuming that all of the peptides encompassing the minimal 1-16 amino acidic sequence display a copper coordination mode that involves three histidines (His(6), His(13), and His(14)). In zinc-Abeta complexes, despite the fact that the metal coordination appears to be more sensitive to solution condition and shows a less rigid geometry around the binding site, a four-histidine coordination mode is seen to be preferred. Lacking a fourth histidine along the Abeta peptide sequence, this geometrical arrangement hints at a Zn(II)-promoted interpeptide aggregation mode.     

Binding of Cu(II) or Zn(II) in a de novo designed triple-stranded alpha-helical coiled-coil toward a prototype for a metalloenzyme.

We previously reported the IZ-3adH peptide, which formed a triple-stranded coiled-coil after binding Ni(II), Cu(II), or Zn(II). In this paper, we report the peptide, IZ-3aH, having a new metal binding specificity. The IZ-3aH peptide was found to bind Cu(II) and Zn(II) and form a triple-stranded coiled-coil. However, it did not bind Ni(II). Metal ion titrations monitored by circular dichroism revealed that the dissociation constants, K(d) were 9 microm for Zn(II) and 10 microm for Cu(II). The bound Cu(II) ion has a planar tetragonal geometry, where the coordination positions are three nitrogens of the His residues and one H(2)O.     

Carbonic Anhydrase Inhibitors. Metal Complexes of 5-(2-Chlorophenyl)-1, 3, 4-Thiadiazole-2-Sulfonamide with Topical Intraocular Pressure Lowering Properties: The Influence of Metal Ions Upon the Pharmacological Activity

Metal complexes of a sulfonamide possessing strong carbonic anhydrase (CA) inhibitory properties, 5-(2-chlorophenyl)-1, 3, 4-thiadiazole-2-sulfonamide (chlorazolamide) have been obtained from the sodium salt of the sulfonamide and the following metal ions: Mg(II), Zn(II), Mn(II), Cu(II), Co(II), Ni(II), Be(II), Cd(II), Pb(II), AI(III), Fe(III) and La(III). The original sulfonamide and its complexes were assayed for the in vitro inhibition of three CA isozymes, CA I, II, and IV, some of which play a critical role in ocular fluid secretion. All these compounds (the sulfonamide and its metal complexes) behaved as powerful inhibitors against the three investigated isozymes. The parent sulfonamide possessed an extremely weak topical pressure lowering effect when administered as a 1-2% suspension into the rabbit eye, but some of its metal complexes, such as the Mg(II), Zn(II), Mn(II) and Cu(II) derivatives, lower intraocular pressure (IOP) in experimental animals very well. Ex vivo data showed a 99.5-99.9% CA II inhibition in ocular fluids and tissues of rabbits treated with these agents, proving that the observed IOP lowering is due to CA inhibition. The influence of the different metal ions upon the efficiency of the obtained complexes as pressure lowering drugs are discussed, leading to the possibility of designing more selective; potent pharmacological agents from this class     

Equilibrium and structural studies on metal complexes of carbohyrates and their derivatives

A summary is presented of the studies of our group on metal complexes of carbohydrates (aldoses, ketoses, mono-, di- and polysaccharides) and their derivatives (aldonic, alduronic acids, polyalcohols, amino sugars, amino acid sugar adducts, AMP, ATP, etc.). The results are reported of equilibrium, electrochemical, solution and solid-state structural studies of complexes of transition metals [Cu(II), Fe(III), Ni(II), Zn(II), Co(II), Ag(I), Mn in different oxidation states and organotin(IV)]. The structural parameters (coordination number, bond distance, and Debye-Waller factor) obtained by extended X-ray absorption fine structure spectroscopic (EXAFS) spectroscopy are discussed in detail. The general rules concerning the formation and structure of such complexes are emphasized.     

IR and Raman study on the interactions of the 5′-GMP and 5′-CMP phosphate groups with Mg(II), Ca(II), Sr(II), Ba(II), Cr(III), Co(II), Cu(II), Zn(II), Cd(II), Al(III) and Ga(III)

The chief motive behind this research is the interest provoked by the presence of metal ions as necessary stabilizers of the negative charges of phosphate groups in nucleic acids. The effect that the presence of different metal ions produces on the band principally assigned to the nu(s) PO(3)(2-) mode has been studied using FT-IR and FT-Raman spectroscopy. The results obtained reveal the diagnostic capacity of these techniques in determining the type of metal ion interaction with respect to the mononucleotides that form DNA and RNA, providing a tool for improving the knowledge of the stabilizing or destabilizing effects of these ions on such macromolecules. The metal complexes of the ribonucleotides 5'-CMP and 5'-GMP with Mg(II), Ca(II), Sr(II), Ba(II), Cr(III), Co(II), Cu(II), Zn(II), Cd(II), Al(III) and Ga(III) were obtained in this study. After studying and analyzing the IR and Raman spectra of all these complexes and comparing them with the spectra of the corresponding disodium salts, it was verified that, independently of the type of nucleotide involved, the presence of the metal in the vicinity of the phosphate group produces an alteration in the aforementioned nu(s) PO(3)(2-) band. This effect is related to the type of interaction that the phosphate group has with the metal. Three components are observed: (1) one near 983-975 cm(-1) (detectable in IR and Raman), associated with phosphate groups in an electrostatic type of interaction with the metal ion, separated by two or more water molecules; (2) another near 989-985 cm(-1) (only in IR), associated with phosphate groups in indirect interaction through the water molecules of the coordination sphere of the metal ions; and (3) the IR and Raman bands near 1014-1001 cm(-1), which represent phosphate groups directly bonded to the metal ion. These results are supported by the behavior of 5'-CMP in aqueous solution in the presence of Mg(II) ions.     

Spectral and thermodynamic properties of Ag(I), Au(III), Cd(II), Co(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(IV), and Zn(II) binding by methanobactin from Methylosinus trichosporium OB3b

Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV–visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ.     

Real-time kinetics of discontinuous and highly conformational metal-ion binding sites of prion protein

The prion protein (PrP) is a metalloprotein with an unstructured region covering residues 60–91 that bind two to six Cu(II) ions cooperatively. Cu can bind to PrP regions C-terminally to the octarepeat region involving residues His111 and/or His96. In addition to Cu(II), PrP binds Zn(II), Mn(II) and Ni(II) with binding constants several orders of magnitudes lower than those determined for Cu. We used for the first time surface plasmon resonance (SPR) analysis to dissect metal binding to specific sites of PrP domains and to determine binding kinetics in real time. A biosensor assay was established to measure the binding of PrP-derived synthetic peptides and recombinant PrP to nitrilotriacetic acid chelated divalent metal ions. We have identified two separate binding regions for binding of Cu to PrP by SPR, one in the octarepeat region and the second provided by His96 and His111, of which His96 is more essential for Cu coordination. The octarepeat region at the N-terminus of PrP increases the affinity for Cu of the full-length protein by a factor of 2, indicating a cooperative effect. Since none of the synthetic peptides covering the octarepeat region bound to Mn and recombinant PrP lacking this sequence were able to bind Mn, we propose a conformational binding site for Mn involving residues 91–230. A novel low-affinity binding site for Co(II) was discovered between PrP residues 104 and 114, with residue His111 being the key amino acid for coordinating Co(II). His111 is essential for Co(II) binding, whereas His96 is more important than His111 for binding of Cu(II).