Katalin Selmeczi

Synthetic models of the active site of catechol oxidase: mechanistic studies

The ability of copper proteins to process dioxygen at ambient conditions has inspired numerous research groups to study their structural, spectroscopic and catalytic properties. Catechol oxidase is a type-3 copper enzyme usually encountered in plant tissues and in some insects and crustaceans. It catalyzes the conversion of a large number of catechols into the respective o-benzoquinones, which subsequently auto-polymerize, resulting in the formation of melanin, a dark pigment thought to protect a damaged tissue from pathogens. After the report of the X-ray crystal structure of catechol oxidase a few years earlier, a large number of publications devoted to the biomimetic modeling of its active site appeared in the literature. This critical review (citing 114 references) extensively discusses the synthetic models of this enzyme, with a particular emphasis on the different approaches used in the literature to study the mechanism of the catalytic oxidation of the substrate (catechol) by these compounds. These are the studies on the substrate binding to the model complexes, the structure-activity relationship, the kinetic studies of the catalytic oxidation of the substrate and finally the substrate interaction with (per)oxo-dicopper adducts. The general overview of the recognized types of copper proteins and the detailed description of the crystal structure of catechol oxidase, as well as the proposed mechanisms of the enzymatic cycle are also presented.

Catecholase activity of a μ-hydroxodicopper(II) macrocyclic complex: structures, intermediates and reaction mechanism

The monohydroxo-bridged dicopper(II) complex (1), its reduced dicopper(I) analogue (2) and the trans-μ-1,2-peroxo-dicopper(II) adduct (3) with the macrocyclic N-donor ligand [22]py4pz (9,22-bis(pyridin-2′-ylmethyl)-1,4,9,14,17,22,27,28,29,30- decaazapentacyclo -[22.2.114,7.111,14.117,20]triacontane-5,7(28),11(29),12,18,20(30), 24(27),25-octaene), have been prepared and characterized, including a 3D structure of 1 and 2. These compounds represent models of the three states of the catechol oxidase active site: met, deoxy (reduced) and oxy. The dicopper(II) complex 1 catalyzes the oxidation of catechol model substrates in aerobic conditions, while in the absence of dioxygen a stoichiometric oxidation takes place, leading to the formation of quinone and the respective dicopper(I) complex. The catalytic reaction follows a Michaelis–Menten behavior. The dicopper(I) complex binds molecular dioxygen at low temperature, forming a trans-μ-1,2-peroxo-dicopper adduct, which was characterized by UV–Vis and resonance Raman spectroscopy and electrochemically. This peroxo complex stoichiometrically oxidizes a second molecule of catechol in the absence of dioxygen. A catalytic mechanism of catechol oxidation by 1 has been proposed, and its relevance to the mechanisms earlier proposed for the natural enzyme and other copper complexes is discussed.

Alkylation of human hemoglobin A0 by the antimalarial drug artemisinin.

In vitro, the heme cofactor of human iron(II) hemoglobin was efficiently and quickly alkylated at meso positions by the peroxide‐based antimalarial drug artemisinin, leading to heme–artemisinin‐derived covalent adducts. This reaction occurred in the absence of any added protease or in the presence of an excess of an extra non‐heme protein, or even when artemisinin was added to hemolysed human blood. This activation of artemisinin by the heme moiety of non‐digested hemoglobin clearly indicates the high affinity of this drug for heme, and its efficient alkylating ability under very mild conditions.

The role of terminal amino group and histidine at the fourth position in the metal ion binding of oligopeptides revisited: Copper(II) and nickel(II) complexes of glycyl-glycyl-glycyl-histamine and its N-Boc protected derivative

Copper(II) and nickel(II) binding properties of two pseudo tetrapeptides, N-Boc-Gly-Gly-Gly-Histamine (BGGGHa) and Gly-Gly-Gly-Histamine (GGGHa) have been investigated by pH-potentiometric titrations, UV-visible-, EPR-, NMR- and ESI-HRMS (electrospray ionization high resolution MS) spectroscopies, in order to compare the role of N-terminal amino group and imidazole moiety at the fourth position in the complex formation processes. Substantially higher stabilities were determined for the ML complexes of GGGHa, compared to those of BGGGHa, supporting the coordination of the terminal amino group and the histamine imidazole of the non-protected ligand. A dimeric Cu(2)H(-2)L(2) species, formed through the deprotonation of peptide groups of the ligands, was found in the GGGHa-copper(II) system. Deprotonation and coordination of further amide nitrogens led to CuH(-2)L and, above pH~10, CuH(-3)L. Experimental data supports a {NH(2), 2 × N(amide),N(im)} macrochelate structure in CuH(-2)L whereas a {NH(2), 3 × N(amide)} coordination environment in CuH(-3)L. The first two amide deprotonation processes were found to be strongly cooperative with nickel(II) and spectroscopic studies proved the transformation of the octahedral parent complexes to square planar, yellow, diamagnetic species, NiH(-2)L and above pH~9, NiH(-3)L. In the basic pH-range deprotonation and coordination of the amide groups also took place in the BGGGHa containing systems, leading to complexes with a {3 × N(amide),N(im)} donor set, and in parallel the re-dissolving of precipitate. Above pH~11, a further proton release from the pyrrolic NH group of the imidazole ring of BGGGHa occurred providing an additional proof for the different binding modes of the two ligands.

Tuning the coordination properties of multi-histidine peptides by using a tripodal scaffold: solution chemical study and catechol oxidase mimicking

Two new tripodal peptides containing non-protected N-terminal (L1, tren3his) and C-terminal (L2, nta3his) histidines have been synthesized in order to combine the structuring effect of tripodal scaffolds and the strong metal binding properties of histidine moieties. In the present work the copper(II) complexes of these ligands have been studied by combined pH-metric, UV-Vis, CD, EPR and MS methods. At a 1 : 1 metal-to-ligand ratio the two ligands behave as the corresponding dipeptides containing N/C-terminal histidines, but above pH 9 the participation of the tertiary amine in the fused chelate rings results in unique binding modes in the case of both ligands. Besides, the formation of oligonuclear complexes also confirms the positive influence of tripodal platforms on metal coordination, and provides the potential to be efficient functional models of oxidase enzymes. Accordingly, the oligonuclear complexes of both ligands exhibit considerable catecholase-like activity. The oxidation of 3,5-di-tert-butyl-catechol proceeds with the participation of separated Cu2+ centers in the presence of L1 complexes. However, the proximity of the two metal ions in the dinuclear complexes of L2 allows their cooperation along the catalytic cycle. Substrate binding modes, effects of reactants, intermediate and side product formation have also been studied, allowing us to propose a plausible catalytic mechanism for each copper(II)–ligand system.

Click synthesis of symmetric bis-triazol ligands and full characterisation of their copper(II)-complexes

Eight novel ligands were prepared from a known symmetric diaza-18-crown-6 (cyclic ligand) and two commercial N,N′-dimethyl-alkyl diamines (acyclic ligands) via the Cu(I)-catalyzed Huisgen dipolar cycloaddition. All C2-symmetric isolated ligands readily formed stable crystalline 1 : 1-copper(II) complexes with cupric perchlorate. Their structural, electrochemical and physico-chemical properties were fully investigated with the help of X-ray diffraction, cyclic voltammetry, FT-IR, UV-visible, and electron paramagnetic resonance (EPR) spectroscopies. Planar – or nearly planar – arrangement of the two N3-triazole nitrogens and the two tertiary amine pivot nitrogens was found in one single four-coordinated species, in four five-coordinated species, and three six-coordinated species, with one or two solvent molecule(s), or two oxygen atoms of the crown ether, occupying the axial position(s) in the solid sate. The electron-donating or electron-withdrawing effect of the N1-substituent on the triazol was found to influence the Cu(II)/Cu(I) redox potential of all studied complexes in DMF. The EPR-spectrum of cyclic complexes in frozen DMF at 100 K exhibited two mononuclear species, one of them likely promoting the formation of dinuclear species as a minor component, whereas most acyclic complex spectra were quite similar.

Nickel(II)-Dipeptidoamine-Based Tetrameric Complex: Structural Study in Solution and in Solid State

The coordination structure of M(4)L(4)H(-8) macromolecules (M = Ni(II), Cu(II), Pd(II)) containing small peptidic ligands (L = Xaa-His or Xaa-His-Yaa) has been predicted primarily on the basis of spectroscopic and potentiometric data in the literature. In this work, the neutral tetranuclear nickel(II) complex 1 formed with four double-deprotonated ligands (L = α-methyl-alanyl-histamine) was prepared, and its crystal structure was determined (C(36)H(56)N(16)Ni(4)O(4)·4.5CH(3)OH·1.5H(2)O: a = 11.2645(4) A, b = 23.5003(8) A, c = 20.9007(7) A, β = 102.321(1)°, monoclinic, P2(1)/c, Z = 4). In complex 1, the metal ions have a square planar geometry with 4N donor set consisting of the N-terminal amino nitrogen, the deprotonated amide nitrogen, the imidazole N(3) atom, and the deprotonated imidazole N(1) atom of the adjacent ligand. The latter nitrogen atom provides the connection of the four NiLH(-2) units forming a C(1) symmetrical saddle-like shape. The complexation of L with Ni(II) ion has been studied by a potentiometric method combined with UV-visible spectrophotometric titration. At pH 8.0, the predominant species is M(4)L(4)H(-8) with pK(4)(oligomerization) = 5.73. The tetranuclear structure of complex 1 was also studied in solution by (1)H and (13)C NMR spectroscopy suggesting a structure of symmetry S(4). DFT calculations on optimized structure in symmetry C(1) and S(4) have been performed to explain the observed differences in solution and in solid state. The nuclearity was also confirmed in solution by ESI-HRMS analysis.

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 {Nim/2Nim,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.

SPR screening of metal chelating peptides in a hydrolysate for their antioxidant properties

There is a growing need in the industrial sector (health, nutrition and cosmetic) to discover new biomolecules with various physico-chemical and bioactive properties. Various beneficial effects of peptides - notably those produced from protein hydrolysis - are reported in the literature. The antioxidant activity involves various mechanisms, among them metal chelation, studied by UV-visible spectrophotometry. In this paper, we set up an original method of screening metal chelating peptides in a hydrolysate using Surface Plasmon Resonance (SPR) for their antioxidant properties. To date, the empirical approach used several cycles of hydrolysate fractionation and bioactivity evaluation until the isolation of the pure bioactive molecule and its identification. Besides, the detection of metal-chelating peptide is not sensitive enough by spectrophotometry. For the first time, metal chelating peptides were screened in hydrolysates using SPR and a correlation was established between affinity constant determined in SPR and metal chelation capacity determined from UV-visible spectrophotometry.

C-Functionalized chiral dioxocyclam and cyclam derivatives with 1,2,3-triazole units: synthesis, complexation properties and crystal structures of copper(II) complexes

New C-functionalized syn- and anti-dioxocyclam and cyclam derivatives with 1,2,3-triazole units attached to carbon atoms within the skeleton were designed as valuable bifunctional chelating agents for applications in nuclear medicine. These macrocyclic chelators were prepared via a multi-step sequence involving α- and β-amino acids, and their copper(II) complexation properties were evaluated. A solution structure in which the triazoles are in axially coordinating positions was proposed for the [Cu(anti-27)]2+ complex. Promising results have been obtained regarding the complexation kinetics (<10 s) and the pseudo-first order half-life for acid-assisted dissociation (t1/2 = 3.21 d, 5 M HCl, 50 °C).