New dioxadiaza- and trioxadiaza-macrocycles containing one dibenzofuran unit with two amino pendant arms: synthesis, protonation and complexation studies

New dioxadiaza- and trioxadiaza-macrocycles containing one rigid dibenzofuran unit (DBF) and N-(2-aminoethyl) pendant arms were synthesized, N,N'-bis(2-aminoethyl)-[17](DBF)N(2)O(2) (L(1)) and N,N'-bis(2-aminoethyl)-[22](DBF)N(2)O(3) (L(2)), respectively. The binding properties of both macrocycles to metal ions and structural studies of their metal complexes were carried out. The protonation constants of both compounds and the stability constants of their complexes with Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), and Pb(2+) were determined at 298.2 K, in aqueous solutions, and at ionic strength 0.10 mol dm(-3) in KNO(3). Mononuclear complexes with both ligands were formed, and dinuclear complexes were only found for L(2). The thermodynamic binding affinities of the metal complexes of L(2) are lower than those of L(1) as expected, but the Pb(2+) complexes of both macrocycles exhibit close stability constant values. On the other hand, the binding affinities of Cd(2+) and Pb(2+) for L(1) are very high, when compared to those of Co(2+), Ni(2+) and Zn(2+). These interesting properties were explained by the presence of the rigid DBF moiety in the backbone of the macrocycle and to the special match between the macrocyclic cavity size and the studied larger metal ions. To elucidate the adopted structures of complexes in solution, the nickel(II) and copper(II) complexes with both ligands were further studied by UV-vis-NIR spectroscopy in DMSO-H(2)O 1 : 1 (v/v) solution. The copper(II) complexes were also studied by EPR spectroscopy in the same mixture of solvents. The crystal structure of the copper complex of L(1) was also determined. The copper(II) displays an octahedral geometry, the four nitrogen atoms forming the equatorial plane and two oxygen atoms, one from the DBF unit and the other one from the ether oxygen, in axial positions. One of the ether oxygens of the macrocycle is out of the coordination sphere. Our results led us to suggest that this geometry is also adopted by the Co(2+) to Zn(2+) complexes, and only the larger Cd(2+) and Pb(2+) manage to form complexes with the involvement of all the oxygen atoms of the macrocyclic backbone.     

Different binding thermodynamics of Ni2+, Cu2+, and Zn2+ to bacitracin A1 determined by capillary electrophoresis

Thermodynamics of the binding of Ni(2+), Cu(2+) and Zn(2+) to bacitracin A(1) was studied by capillary electrophoresis measuring the peptide effective mobility at different pH in the presence of increasing concentration of the three ligands. The affinity follows the order Ni(2+) > Cu(2+) > Zn(2+), with association constant values of (2.3 +/- 0.1)x10(4), (4.9 +/- 0.2)x10(3), and (1.5 +/- 0.1)x10(3) M(-1), respectively. The only model able to rationalize mobility data implies that metal ion binds to the P(0) peptide form. Moreover, mobility values indicated a change of bacitracin A(1) acidic properties on Ni(2+) and Cu(2+) binding, with a shift of the pK(a) of N-terminal Ile-1 from 7.6 to about 5 and of the pK(a) of the delta-amino group of D-Orn-7 from 9.7 to about 7. Even though on Zn(2+) binding a shift of the N-terminal Ile-1 pK(a) was observed, restrictions in the pH range suitable for investigation, due to precipitation phenomena, did not allow establish if the shift of D-Orn-7 lateral chain pK(a) also occurred. Nonetheless, if present, the shift should be limited to the 7.8-9.7 range. Mobility data indicated that the Stokes radius of the complexes is ca. 3 A lower than that of the free peptide. The present results indicate that metal-ion binding to bacitracin A(1) is more complex than previously assumed.     

Polythiol binding to biologically relevant metal ions

The coordination properties of three peptides with CXXC motif: Ac-GCASCDNCRACKK-NH(2), Ac-GCASCDNCRAAKK-NH(2) and Ac-GCASCDNARAAKK-NH(2) as donors of four, three and two thiol ligands for Ni(2+),Cd(2+), Zn(2+) and Bi(3+) were studied by potentiometric titrations, UV-Vis and CD spectra measurements. Since the stability of the complexes is closely connected with the amount of the metal-bound cysteine sulfurs, competition plots of the complexes of peptides with 2, 3 and 4 cysteines further prove the involvement of all thiols in the metal ion binding. Furthermore, the sulfur-bound zinc complexes appear to be much more stable than the sulfur-bound nickel ones. The stabilities of the studied complexes decreases in the series Bi(3+) ? Cd(2+) > Zn(2+) > Ni(2+).     

Metal-ion binding and limited proteolysis of betabellin 15D, a designed beta-sandwich protein

Betabellin 15D is a 64-residue, disulfide-bridged homodimer. When folded into a beta structure, the protein is predicted to have two clusters of three histidine residues, each cluster able to bind a divalent metal ion. When the protein was incubated with Cu2+, Zn2+, Co2+, or Mn2+, metal complexes of betabellin 15D were observed by electrospray-ionization mass spectrometry. The relative abundances of the ionic complexes suggested an order of affinities of Cu2+ > Zn2+ > Co2+ > Mn2+, consistent with solution-phase affinities for nitrogen- or sulfur-containing ligands. Limited proteolysis of betabellin 15D by immobilized pepsin, as measured by nanoelectrospray-ionization mass spectrometry, showed that the Phe12-Ser13 peptide bond of betabellin 15D was cleaved more slowly in the presence of Cu2+ than in its absence. Because Cu2+ has little or no effect on the catalytic rate of pepsin, the slower cleavage of the Phe12-Ser13 peptide bond may be due to its decreased accessibility caused by Cu(2+)-induced folding of betabellin 15D.     

Cyclic and hairpin peptide complexes of heme

We have synthesized and characterized a new class of heme-peptide complexes using disulfide-linked hairpin-turn and cyclic peptides and compared these to their linear analogues. The binding affinities, helicities, and mechanism of binding of linear, hairpin, and cyclic peptides to [FeIII(coproporphyrin-I)]+ have been determined. In a minimalist approach, we utilize amphiphilic peptide sequences (15-mers), where a central histidine provides heme ligation, and the hydrophobic effect is used to optimize heme-peptide complex stability. We have incorporated disulfide bridges between amphiphilic peptides to make hairpin and even cyclic peptides that bind heme extremely well, roughly 5 x 106 times more strongly than histidine itself. CD studies show that the cyclic peptide heme complexes are completely alpha-helical. NMR spectra of paramagnetic complexes of the peptides show that the 15-mer peptides bind sequentially, with an observable monopeptide, high-spin intermediate. In contrast, the cyclic peptide complexes ligate both imidazoles cooperatively to the heme, producing only a low-spin complex. Electrochemical measurements of the E1/2 of the FeIII(coproporphyrin-I)+ complexes of these peptides are all at fairly low potentials, ranging from -215 to -252 mV versus NHE at pH 7.     

Aminoethylglycine-functionalized Ru(bpy)3(2+) with pendant bipyridines self-assemble multimetallic complexes by copper and zinc coordination

Directed self-assembly using inorganic coordination chemistry is an attractive approach for making functional supramolecular structures. In this article, the synthesis and characterization of Ru(bpy) 3 (2+) compounds derivatized with aminoethylglycine (aeg) substituents containing pendant bipyridine (bpy) ligands is presented. The free bpy ligands in these complexes are available for metal chelation to form coordinative cross-links; addition of Cu (2+) or Zn (2+) assembles heterometallic structures containing two or three transition-metal complexes. Control over relative placement of metal complexes is accomplished using two strategies: two bipyridine-containing aeg strands tethered to Ru(bpy) 3 (2+) allow intramolecular coordination and result in a dimetallic hairpin motif. Ru(bpy) 3 (2+) modified with a single strand forms intermolecular cross-links forming the trimetallic complex. Each of these is characterized by a range of methods, and their photophysical properties are compared. These data, and comparison to an acetyl aeg- modified Ru(bpy) 3 (2+) complex, confirm that the metal ions cross-link bpy-containing aeg strands. Heterometallic complexes containing bound Cu (2+) cause a dramatic reduction in the Ru(bpy) 3 (2+) quantum yields and lifetimes. In contrast, the Ru(bpy) 3 (2+) hairpin with coordinated Zn (2+) has only a slight decrease in quantum yield but no change in lifetime, which could be a result of steric impacts on structure in the dimetallic species. Analogous effects are not observed in the trimetallic Ru-Zn-Ru structures in which this constraint is absent. Each of these heterometallic structures represents a facile and reconfigurable means to construct multimetallic structures by metal-coordination-based self-assembly of modular artificial peptide units.     

Toward optimized high-relaxivity MRI agents: thermodynamic selectivity of hydroxypyridonate/catecholate ligands

The thermodynamic selectivity for Gd(3+) relative to Ca(2+), Zn(2+), and Fe(3+) of two ligands of potential interest as magnetic resonance imaging (MRI) contrast agents has been determined by NMR spectroscopy and potentiometric and spectrophotometric titration. The two hexadentate ligands TREN-6-Me-3,2-HOPO (H(3)L2) and TREN-bisHOPO-TAM-EA (H(4)L3) incorporate 2,3-dihydroxypyridonate and 2,3-dihydroxyterephthalamide moieties. They were chosen to span a range of basicity while maintaining a structural motif similar to that of the parent ligand, TREN-1-Me-3,2-HOPO (H(3)L1), in order to investigate the effect of the ligand basicity on its selectivity. The 1:1 stability constants (beta(110)) at 25 degrees C and 0.1 M KCl are as follows. L2: Gd(3+), 20.3; Ca(2+), 7.4; Zn(2+), 11.9; Fe(3+), 27.9. L3: Gd(3+), 24.3; Ca(2+), 5.2; Zn(2+), 14.6; Fe(3+), 35.1. At physiological pH, the selectivity of the ligand for Gd(3+) over Ca(2+) increases with the basicity of the ligand and decreases for Gd(3+) over Fe(3+). These trends are consistent with the relative acidities of the various metal ions;- more basic ligands favor harder metals with a higher charge-to-radius ratio. The stabilities of the Zn(2+) complexes do not correlate with basicity and are thought to be more influenced by geometric factors. The selectivities of these ligands are superior to those of the octadentate poly(aminocarboxylate) ligands that are currently used as MRI contrast agents in diagnostic medicine.     

Pyridine-based lanthanide complexes combining MRI and NIR luminescence activities

A series of novel triazole derivative pyridine-based polyamino-polycarboxylate ligands has been synthesized for lanthanide complexation. This versatile platform of chelating agents combines advantageous properties for both magnetic resonance (MR) and optical imaging applications of the corresponding Gd(3+) and near-infrared luminescent lanthanide complexes. The thermodynamic stability constants of the Ln(3+) complexes, as assessed by pH potentiometric measurements, are in the range log K(LnL)=17-19, with a high selectivity for lanthanides over Ca(2+), Cu(2+), and Zn(2+). The complexes are bishydrated, an important advantage to obtain high relaxivities for the Gd(3+) chelates. The water exchange of the Gd(3+) complexes (k(ex)(298)=7.7-9.3×10(6) s(-1)) is faster than that of clinically used magnetic resonance imaging (MRI) contrast agents and proceeds through a dissociatively activated mechanism, as evidenced by the positive activation volumes (ΔV(≠)=7.2-8.8 cm(3) mol(-1)). The new triazole ligands allow a considerable shift towards lower excitation energies of the luminescent lanthanide complexes as compared to the parent pyridinic complex, which is a significant advantage in the perspective of biological applications. In addition, they provide increased epsilon values resulting in a larger number of emitted photons and better detection sensitivity. The most conjugated system PheTPy, bearing a phenyl-triazole pendant on the pyridine ring, is particularly promising as it displays the lowest excitation and triplet-state energies associated with good quantum yields for both Nd(3+) and Yb(3+) complexes. Cellular and in vivo toxicity studies in mice evidenced the non-toxicity and the safe use of such bishydrated complexes in animal experiments. Overall, these pyridinic ligands constitute a highly versatile platform for the simultaneous optimization of both MRI and optical properties of the Gd(3+) and the luminescent lanthanide complexes, respectively.     

Doubly charged protonated a ions derived from small peptides

Protonated a(2) and a(3) (therefore doubly charged) ions in which both charges lie on the peptide backbone are formed in collision-induced dissociations of [La(III)(peptide)(CH(3)CN)(m)](3+) complexes. Abundant (a(3)+H)(2+) ions are formed from triproline (PPP) and peptides with a proline residue at the N-terminus; these peptides are the most effective in producing ions of the type (a(2)+H)(2+) and (a(3)+H)(2+). A systematic study of the effect of the location of the proline residue and other residues of aliphatic amino acids on the generation of protonated a ions is reported. Density functional theory calculations at B3LYP/6-311++G(d,p) gave the proton affinity of the a(3) ion derived from PPP to be 167.6 kcal mol(-1), 2.6 kcal mol(-1) higher than that of water. The protonated a(2) ions of diglycine and diproline and a(3) ions of triglycine have lower proton affinities and are only observed in lower abundances, possibly due to proton transfer to water in ion-molecule reactions.     

A spectrochemical walk: single-site perturbation within a series of six-coordinate ferrous complexes

A series of ferrous complexes with the pentadentate ligand 2,6-(bis-(bis-2-pyridyl)methoxymethane)pyridine (PY5) was prepared and examined. PY5 binds ferrous iron in a square-pyramidal geometry, leaving a single coordination site accessible for complexation of a wide range of monodentate exogenous ligands: [Fe(II)(PY5)(X)](n+), X = MeOH, H(2)O, MeCN, pyridine, Cl-, OBz-, N(3)-, MeO-, PhO-, and CN-. The spin-states of these ferrous complexes are extremely sensitive to the nature of the single exogenous ligand; the spectroscopic and structural properties correlate with their high-spin (hs) or low-spin (ls) electronic ground state. Systematic metrical trends within six crystallographic structures clearly indicate a preferred conformational binding mode of the PY5 ligand. The relative binding affinities of the exogenous ligands in MeOH indicate that exogenous ligand charge is the primary determinant of the binding affinity; the [Fe(II)(PY5)](2+) unit preferentially binds anionic ligands over neutral ligands. At parity of charge, strong-field ligands are preferentially bound over weak-field ligands. In MeOH, the pK(a) of the exogenously ligated MeOH in [Fe(PY5)(MeOH)](2+) (9.1) limits the scope of exogenous ligands, as strongly basic ligands preferentially deprotonate [Fe(PY5)(MeOH)](2+) to yield [Fe(PY5)(OMe)](1+) rather than ligate to the ferrous center. Exogenous ligation by a strongly basic ligand, however, can be achieved in polar aprotic solvents.     

Modeling Structural Coordination and Ligand Binding in Zinc Proteins with a Polarizable Potential

As the second most abundant cation in human body, zinc is vital for the structures and functions of many proteins. Zinc-containing matrix metalloproteinases (MMPs) have been widely investigated as potential drug targets in a range of diseases ranging from cardiovascular disorders to cancers. However, it remains a challenge in theoretical studies to treat zinc in proteins with classical mechanics. In this study, we examined Zn(2+) coordination with organic compounds and protein side chains using a polarizable atomic multipole based electrostatic model. We find that polarization effect plays a determining role in Zn(2+) coordination geometry in both matrix metalloproteinase (MMP) complexes and in zinc-finger proteins. In addition, the relative binding free energies of selected inhibitors binding with MMP13 have been estimated and compared with experimental results. While not directly interacting with the small molecule inhibitors, the permanent and polarizing field of Zn(2+) exerts a strong influence on the relative affinities of the ligands. The simulation results also reveal the polarization effect on binding is ligand dependent and thus difficult to be incorporated into fixed-charge models implicitly.     

Electrospray-ionization mass spectrometry for the detection of discrete peptide/metal-ion complexes involving multiple cysteine (sulfur) ligands.

Conditions have been developed to characterize the reversible interaction of one or more Zn(II) ions with cysteine (sulfur) ligands on metal-binding peptides by electrospray-ionization (ES) mass spectrometry. A 71-residue peptide with two separate clusters of four cysteine residues was selected as a model to optimize both the solution and electrospray variables most likely to affect the detection of stable cysteine (sulfur) ligand/Zn interactions. By infusing peptide in water alone, stable electrospray and ion signals were produced in both the absence and presence of up to 100 microM zinc sulfate. In the absence of Zn(II), the calculated mass of the fully reduced peptide (8248.5 Da) was observed (8248.4 +/- 0.4 Da). In the presence of Zn(II), peptides with zero, one and two bound Zn atoms were detected; all three species were present in several different charge states. The overall charge envelope was typically unchanged in the presence of Zn; the charge-state optimum (10+) observed for this peptide was apparently unaffected by the presence of bound Zn. The interaction of Zn(II) ions with sulfur ligands in this peptide appeared to result in tetracoordinate covalent bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bi-1,10-phenanthrolines and their mononuclear Ru(II) complexes

A series of four biphen (phen = 1,10-phenanthroline) ligands, 2,2'-biphen (1), 3,3'-biphen (2), 2,2'-dimethylene-3,3'-biphen (3), and 2,3'-dimethylene-3,2'-biphen (4), is prepared by coupling and Friedl?nder methodology. The corresponding mononuclear Ru(II) complexes, [Ru(1-4)(Mebpy)(2)](2+) where Mebpy = 4,4'-dimethyl-2,2'-bipyridine, are prepared. These complexes show long wavelength electronic absorptions at 441-452 nm and emissions at 622-641 nm. Metal-based oxidations occur in the range 1.18-1.21 V, and ligand-based reductions, at -1.20 to -1.30 V. The addition of Zn(2+), Cd(2+), or Hg(2+) ions results in a strong enhancement and red shift of the luminescence of complex Ru-3. Alkali and alkaline earth metal ions barely affect the luminescence of Ru-3 while transition metal ions such as Co(2+), Cu(2+), Ni(2+), and Mn(2+) lead to efficient quenching of the Ru-3 luminescence. The luminescence of Ru-2 and Ru-4 is quenched in the presence of Zn(2+) because of a conformationally induced reduction in electronic communication between the two phen halves of the ligand. The addition of Zn(2+) has only a slight effect on the luminescence of Ru-1 because of steric hindrance toward complexation.     

Evaluation of the metal binding properties of the histidine-rich antimicrobial peptides histatin 3 and 5 by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) was used to investigate metal ion interactions with salivary peptides histatin 3 (H3) and histatin 5 (H5). Conformational changes of these peptides in the presence of metal ions were studied using circular dichroism spectroscopy. H3 and H5 formed high affinity complexes with Cu(2+) and Ni(2+) and, to a lesser extent, with Zn(2+). Both peptides show the potential for multiple binding sites for Cu(2+) and Ni(2+) and only a single strong binding site for Zn(2+). The binding of a third Cu(2+) ion to H3 seems to enable the binding of a fourth ion to H3. The binding of a second and third Ni(2+) ion to H5 has a similar effect in enabling the binding of a fourth ion. None of the metal ions examined stabilized a regular secondary structure for either peptide. Subtle changes in overall conformation are seen with the addition of Cu(2+) to both H3 and H5.     

Molecular design of specific metal-binding peptide sequences from protein fragments: theory and experiment

A novel strategy is presented for designing peptides with specific metal-ion chelation sites, based on linking computationally predicted ion-specific combinations of amino acid side chains coordinated at the vertices of the desired coordination polyhedron into a single polypeptide chain. With this aim, a series of computer programs have been written that 1) creates a structural combinatorial library containing Z(i)-(X)(n)-Z(j) sequences (n=0-14; Z: amino acid that binds the metal through the side chain; X: any amino acid) from the existing protein structures in the non-redundant Protein Data Bank; 2) merges these fragments into a single Z(1)-(X)(n(1) )-Z(2)-(X)(n(2) )-Z(3)-(X)(n(3) )--Z(j) polypeptide chain; and 3) automatically performs two simple molecular mechanics calculations that make it possible to estimate the internal strain in the newly designed peptide. The application of this procedure for the most M(2+)-specific combinations of amino acid side chains (M: metal; see L. Rulísek, Z. Havlas J. Phys. Chem. B 2003, 107, 2376-2385) yielded several peptide sequences (with lengths of 6-20 amino acids) with the potential for specific binding with six metal ions (Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+) and Hg(2+)). The gas-phase association constants of the studied metal ions with these de novo designed peptides were experimentally determined by MALDI mass spectrometry by using 3,4,5-trihydroxyacetophenone as a matrix, whereas the thermodynamic parameters of the metal-ion coordination in the condensed phase were measured by isothermal titration calorimetry (ITC), chelatometry and NMR spectroscopy methods. The data indicate that some of the computationally predicted peptides are potential M(2+)-specific metal-ion chelators.     

Intrinsic Ca2+ affinities of peptides: Application of the kinetic method to analogs of calcium-binding site III of rabbit skeletal troponin C

We extended the kinetic method to determine the intrinsic affinities of nonvolatile organic molecules with divalent metal ions and then applied the amended method to determine the calcium affinities of peptide analogs of the calcium-binding site III of rabbit skeletal troponin C. Metal-bis(peptide) complexes of the composition ([H2Pi + H2Pii] - H + Ca)+, where H2P is a neutral peptide, were introduced into the gas phase by fast atom bombardment. The extended kinetic method recognizes that the dissociation characteristics of a singly charged, bis(peptide) complexes of divalent metal ions are determined by not only the metal-ion affinity but also the proton affinities of the neutral and deprotonated peptides. The modified method requires one to measure the relative abundances of [H2P - H + Ca]+, [H2P + H]+, and [H2P - H]- ions that form upon collisional activation of mixed peptide/metal complexes, proton-bound peptide dimers, and deprotonated peptide dimers, respectively. We found, by using the modified method, that the set of peptides has a different affinity order than that in solution. Peptides with one aspartic acid have a higher intrinsic Ca2+ affinity than those with two aspartates. The location of the aspartic acid (Asp) residues at various positions also affects the Ca2+ affinity. Those peptides with one Asp in the middle of the chain have higher Ca2+ affinities than those with Asp on the end because the former peptides offer greater polarizability to stabilize the charge. Peptides with two Asp's located in close proximity have higher intrinsic calcium affinities than those with aspartates positioned further apart.     

Mn2+ complexes with 12-membered pyridine based macrocycles bearing carboxylate or phosphonate pendant arm: crystallographic, thermodynamic, kinetic, redox, and 1H/17O relaxation studies

Mn(2+) complexes represent an alternative to Gd(3+) chelates which are widely used contrast agents in magnetic resonance imaging. In this perspective, we investigated the Mn(2+) complexes of two 12-membered, pyridine-containing macrocyclic ligands bearing one pendant arm with a carboxylic acid (HL(1), 6-carboxymethyl-3,6,9,15-tetraazabicyclo[9.3.1] pentadeca-1(15),11,13-triene) or a phosphonic acid function (H(2)L(2), 6-dihydroxyphosphorylmethyl-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene). Both ligands were synthesized using nosyl or tosyl amino-protecting groups (starting from diethylenetriamine or tosylaziridine). The X-ray crystal structures confirmed a coordination number of 6 for Mn(2+) in their complexes. In aqueous solution, these pentadentate ligands allow one free coordination site for a water molecule. Potentiometric titration data indicated a higher basicity for H(2)L(2) than that for HL(1), related to the electron-donating effect of the negatively charged phosphonate group. According to the protonation sequence determined by (1)H and (31)P pH-NMR titrations, the first two protons are attached to macrocyclic amino groups whereas the subsequent protonation steps occur on the pendant arm. Both ligands form thermodynamically stable complexes with Mn(2+), with full complexation at physiological pH and 1:1 metal to ligand ratio. The kinetic inertness was studied via reaction with excess of Zn(2+) under various pHs. The dissociation of MnL(2) is instantaneous (at pH 6). For MnL(1), the dissociation is very fast (k(obs) = 1-12 × 10(3) s(-1)), much faster than that for MnDOTA, MnNOTA, or the Mn(2+) complex of the 15-membered analogue. It proceeds exclusively via the dissociation of the monoprotonated complex, without any influence of Zn(2+). In aqueous solution, both complexes are air-sensitive leading to Mn(3+) species, as evidenced by UV-vis and (1)H NMRD measurements and X-ray crystallography. Cyclic voltammetry gave low oxidation peak potentials (E(ox) = 0.73 V for MnL(1) and E(ox) = 0.68 V for MnL(2)), in accordance with air-oxidation. The parameters governing the relaxivity of the Mn(2+) complexes were determined from variable-temperature (17)O NMR and (1)H NMRD data. The water exchange is extremely fast, k(ex) = 3.03 and 1.77 × 10(9) s(-1) for MnL(1) and MnL(2), respectively. Variable-pressure (17)O NMR measurements have been performed to assess the water exchange mechanism on MnL(1) and MnL(2) as well as on other Mn(2+) complexes. The negative activation volumes for both MnL(1) and MnL(2) complexes confirmed an associative mechanism of the water exchange as expected for a hexacoordinated Mn(2+) ion. The hydration number of q = 1 was confirmed for both complexes by (17)O chemical shifts. A relaxometric titration with phosphate, carbonate or citrate excluded the replacement of the coordinated water molecule by these small endogenous anions.     

The inorganic perspective of nerve growth factor: interactions of Cu2+ and Zn2+ with the N-terminus fragment of nerve growth factor encompassing the recognition domain of the TrkA receptor

There is a significant overlap between brain areas with Zn(2+) and Cu(2+) pathological dys-homeostasis and those in which the nerve growth factor (NGF) performs its biological role. The protein NGF is necessary for the development and maintenance of the sympathetic and sensory nervous systems. Its flexible N-terminal region has been shown to be a critical domain for TrkA receptor binding and activation. Computational analyses show that Zn(2+) and Cu(2+) form pentacoordinate complexes involving both the His4 and His8 residues of the N-terminal domain of one monomeric unit and the His84 and Asp105 residues of the other monomeric unit of the NGF active dimer. To date, neither experimental data on the coordination features have been reported, nor has one of the hypotheses according to which Zn(2+) and Cu(2+) may have different binding environments or the Ser1 α-amino group could be involved in coordination been supported. The peptide fragment, encompassing the 1-14 sequence of the human NGF amino-terminal domain (NGF(1-14)), blocked at the C terminus, was synthesised and its Cu(2+) and Zn(2+) complexes characterized by means of potentiometric and spectroscopic (UV/Vis, CD, NMR, and EPR) techniques. The N-terminus-acetylated form of NGF(1-14) was also investigated to evaluate the involvement of the Ser1 α-amino group in metal-ion coordination. Our results demonstrate that the amino group is the first anchoring site for Cu(2+) and is involved in Zn(2+) coordination at physiological pH. Finally, a synergic proliferative activity of both NGF(1-14) and the whole protein on SHSY5Y neuroblastoma cell line was found after treatment in the presence of Cu(2+). This effect was not observed after treatment with the N-acetylated peptide fragment, demonstrating a functional involvement of the N-terminal amino group in metal binding and peptide activity.     

Effects of transition metal ion identity and π-cation interactions in metal-bis(peptide) complexes containing phenylalanine

Electrospray ionization-tandem mass spectrometry was used to study the effects of the metal ion identity and π-cation interactions on the dissociation pathways of metal-bis(peptide) complexes, where the metal is either Mn(2+), Co(2+), Ni(2+), Cu(2+), or Zn(2+); and the peptide is either FGGF, GGGG, GF, or GG, where G is glycine and F is phenylalanine. The [(FGGF)(FGGF-H) + M(2+)](+) and [(GGGG)(GGGG-H) + M(2+)](+) complexes dissociated by losing one FGGF or GGGG, respectively. Relative binding affinities were measured using the crossover points, where the parent and product ions were equal in ion abundance and a normalized-collision energy scale. The results indicate the relative binding affinities for FGGF and GGGG follow the same order with respect to the transition metal ion identity: Cu(2+) < Ni(2+) < Mn(2+) ≈ Zn(2+) < Co(2+), and the π-cation interactions in the FGGF complex have a measureable stabilizing effect. In contrast, the main fragmentation channels of [(GF)(GF-H) + M(2+)]+ and [(GG)(GG-H) + M(2+)](+) are loss of CO(2) and 2CO(2) with the [(GF)(GF-H) + M(2+)](+) complex also exhibiting cinnamic acid ,GF, residual glycine, cinnamate and styrene loss.