Redox cycling of iron complexes of N-(dithiocarboxy)sarcosine and N-methyl-d-glucamine dithiocarbamate

Iron(II)-dithiocarbamate complexes are used to trap nitrogen monoxide in biological samples, and the resulting nitrosyliron(II)-dithiocarbamate is detected and quantified by ESR. As the chemical properties of these compounds have been little studied, we investigated whether iron dithiocarbamate complexes can redox cycle. The electrode potentials of iron complexes of N-(dithiocarboxy)sarcosine (dtcs) and N-methyl-d-glucamine dithiocarbamate (mgd) are 56 and -25 mV at pH 7.4, respectively, as measured by cyclic voltammetry. The autoxidation and Fenton reaction of iron(II)-dtcs and iron(II)-mgd were studied by stopped-flow spectrophotometry with both iron(II) complexes and dioxygen or hydrogen peroxide in excess. In the case of excess iron(II)-dtcs and -mgd complexes, the rate constants of the autoxidation and the Fenton reaction are (1.6-3.2) x 10(4) and (0.7-1.1) x 10(5) M(-1) s(-1), respectively. In the presence of nitrogen monoxide, the oxidation of iron(II)-dtcs and iron(II)-mgd by hydrogen peroxide is significantly slower (ca. 10-15 M(-1) s(-1)). The physiological reductants ascorbate, cysteine, and glutathione efficiently reduce iron(III)-dtcs and iron(III)-mgd. Therefore, iron bound to dtcs and mgd can redox cycle between iron(II) and iron(III). The ligands dtcs and mgd are slowly oxidized by hydrogen peroxide with rate constants of 5.0 and 3.8 M(-1) s(-1), respectively.     

Redox cycling of iron complexes of N-(dithiocarboxy)sarcosine and N-methyl-D-glucamine dithiocarbamate

Iron(II)-dithiocarbamate complexes are used to trap nitrogen monoxide in biological samples, and the resulting nitrosyliron(II)-dithiocarbamate is detected and quantified by ESR. As the chemical properties of these compounds have been little studied, we investigated whether iron dithiocarbamate complexes can redox cycle. The electrode potentials of iron complexes of N-(dithiocarboxy)sarcosine (dtcs) and N-methyl-d-glucamine dithiocarbamate (mgd) are 56 and -25 mV at pH 7.4, respectively, as measured by cyclic voltammetry. The autoxidation and Fenton reaction of iron(II)-dtcs and iron(II)-mgd were studied by stopped-flow spectrophotometry with both iron(II) complexes and dioxygen or hydrogen peroxide in excess. In the case of excess iron(II)-dtcs and -mgd complexes, the rate constants of the autoxidation and the Fenton reaction are (1.6-3.2) x 10(4) and (0.7-1.1) x 10(5) M(-1) s(-1), respectively. In the presence of nitrogen monoxide, the oxidation of iron(II)-dtcs and iron(II)-mgd by hydrogen peroxide is significantly slower (ca. 10-15 M(-1) s(-1)). The physiological reductants ascorbate, cysteine, and glutathione efficiently reduce iron(III)-dtcs and iron(III)-mgd. Therefore, iron bound to dtcs and mgd can redox cycle between iron(II) and iron(III). The ligands dtcs and mgd are slowly oxidized by hydrogen peroxide with rate constants of 5.0 and 3.8 M(-1) s(-1), respectively.     

Inhibition of the Fenton reaction by nitrogen monoxide

The toxicity of iron is believed to originate from the Fenton reaction which produces the hydroxyl radical and/or oxoiron(2+). The effect of nitrogen monoxide on the kinetics of the reaction of iron(II) bound to citrate, ethylenediamine-N,N′-diacetate (edda), ethylenediamine-N,N,N′,N′-tetraacetate (edta), (N-hydroxyethyl)amine-N,N′,N′-triacetate (hedta), and nitrilotriacetate (nta) with hydrogen peroxide was studied by stopped-flow spectrophotometry. Nitrogen monoxide inhibits the Fenton reaction to a large extent. For instance, hydrogen peroxide oxidizes iron(II) citrate with a rate constant of 5.8×10³ M⁻¹ s⁻¹, but in the presence of nitrogen monoxide, the rate constant is 2.9×10² M⁻¹ s⁻¹ . Similar to hydrogen peroxide, the reaction of tert-butyl hydroperoxide with iron(II) complexes is also efficiently inhibited by nitrogen monoxide. Generally, nitrogen monoxide binds rapidly to a coordination site of iron(II) occupied by water. The rate of oxidation is influenced by the rate of dissociation of the nitrogen monoxide from iron(II). Electronic Supplementary Material Supplementary material is available for this article at     

A re-investigation of the reactions between superoxide anion and metal picolinate complexes

Rate constants for the reactions of superoxide with the α-picolinate ion and its complexes with copper(II), iron(III) and zinc(II), and for the reaction of α-picolinate with the hydrated electron, were measured using pulse radiolysis. The rate constant for the reaction of superoxide with copper(II)picolinate at pH 9 [(4.1 ± 0.4) × 10⁷l mol⁻¹ s⁻¹] was an order of magnitude higher than that determined previously (W. H. Bannister, J. V. Bannister, A. J. F. Searle and P. J. Thornally, Inorg. Chim. Acta, 78, 139 (1983)) using a less direct competitive inhibition method. The corresponding rate constant for iron(III)picolinate [(7.5 ± 1.5) X 10³ l mol⁻¹ s⁻] was an order of magnitude lower than a previous pulse radiolysis determination (same reference as above). We are not able to reconcile these two values for iron(III)picolinate, although a possible source of spuriously high results is contamination with the kinetically active copper(II) complex. The likely roles of iron(III)picolinate and other low molecular weight iron complexes as potential catalysts of an in vivo superoxide-driven Fenton reaction are discussed in the light of present measurements.     

The kinetics of the reaction of nitrogen dioxide with iron(II)- and iron(III) cytochrome c

The reactions of NO₂ with both oxidized and reduced cytochrome c at pH 7.2 and 7.4, respectively, and with N-acetyltyrosine amide and N-acetyltryptophan amide at pH 7.3 were studied by pulse radiolysis at 23 °C. NO₂ oxidizes N-acetyltyrosine amide and N-acetyltryptophan amide with rate constants of (3.1±0.3)×10⁵ and (1.1±0.1)×10⁶ M⁻¹ s⁻¹, respectively. With iron(III)cytochrome c, the reaction involves only its amino acids, because no changes in the visible spectrum of cytochrome c are observed. The second-order rate constant is (5.8±0.7)×10⁶ M⁻¹ s⁻¹ at pH 7.2. NO₂ oxidizes iron(II)cytochrome c with a second-order rate constant of (6.6±0.5)×10⁷ M⁻¹ s⁻¹ at pH 7.4; formation of iron(III)cytochrome c is quantitative. Based on these rate constants, we propose that the reaction with iron(II)cytochrome c proceeds via a mechanism in which 90% of NO₂ oxidizes the iron center directly—most probably via reaction at the solvent-accessible heme edge—whereas 10% oxidizes the amino acid residues to the corresponding radicals, which, in turn, oxidize iron(II). Iron(II)cytochrome c is also oxidized by peroxynitrite in the presence of CO₂ to iron(III)cytochrome c, with a yield of ~60% relative to peroxynitrite. Our results indicate that, in vivo, NO₂ will attack preferentially the reduced form of cytochrome c; protein damage is expected to be marginal, the consequence of formation of amino acid radicals on iron(III)cytochrome c.     

Pre-steady-state kinetic study on the formation of Compound I and II of ligninase

The reaction between ligninase and hydrogen peroxide yielding Compound I has been investigated using a stopped-flow rapid-scan spectrophotometer. The optical absorption spectrum of Compound I appears different to that reported by Andrawis, A. et al. (1987) and Renganathan, V. and Gold, M.H. (1986), in that the Soret-maximum is at 401 nm rather than 408 nm. The second-order rate constant (4.2·10⁵ M⁻¹·s⁻¹) for the formation of Compound I was independent of pH (pH 3.0–6.0). In the absence of external electron donors, Compound I decayed to Compound II with a half-life of 5–10 s at pH 3.1. The rate of this reaction was not affected by the H₂O₂ concentration used. In the presence of either veratryl alcohol or ferrocyanide, Compound II was rapidly generated. With ferrocyanide, the second-order rate constant increased from 1.9·10⁴ M⁻¹·s⁻¹ to 6.8·10⁶ M⁻¹·s⁻¹ when the pH was lowered from 6.0 to 3.1. With veratryl alcohol as an electron donor, the second-order rate constant for the formation of Compound II increased from 7.0·10³ M⁻¹·s⁻¹ at pH 6.0 to 1.0·10⁵ M⁻¹·s⁻¹ at pH 4.5. At lower pH values the rate of Compound II formation no longer followed an exponential relationship and the steady-state spectral properties differed to those recorded in the presence of ferrocyanide. Our data support a model of enzyme catalysis in which veratryl alcohol is oxidized in one-electron steps and strengthen the view that veratryl alcohol oxidation involves a substrate-modified Compound II intermediate which is rapidly reduced to the native enzyme.     

Quinolinic acid — Iron(II) complexes: Slow autoxidation,but enhanced hydroxyl radical production in the Fenton reaction

Quinolinate (pyridine-2,3-dicarboxylic acid, Quin) is a neurotoxic tryptophan metabolite produced mainly by immune-activated macrophages. It is implicated in the pathogenesis of several brain disorders including HIV-associated dementia. Previous evidence suggests that Quin may exert its neurotoxic effects not only as an agonist on the NMDA subtype of glutamate receptor, but also by a receptor-independent mechanism. In this study we address ability of ferrous quinolinate chelates to generate reactive oxygen species. Autoxidation of Quin-Fe(II) complexes, followed in Hepes buffer at pH 7.4 using ferrozine as the Fe(II) detector, was found to be markedly slower in comparison with iron unchelated or complexed to citrate or ADP. The rate of Quin-Fe(II) autoxidation depends on pH (squared hydroxide anion concentration), is catalyzed by inorganic phosphate, and in both Hepes and phosphate buffers inversely depends on Quin concentration. These observations can be explained in terms of anion catalysis of hexaaquairon(II) autoxidation, acting mainly on the unchelated or partially chelated pool of iron. In order to follow hydroxyl radical generation in the Fenton chemistry, electron paramagnetic resonance (EPR) spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was employed. In the mixture consisting of 100 mM DMPO, 0.1 mM Fe(II), and 8.8 mM hydrogen peroxide in phosphate buffer pH 7.4, 0.5 mM Quin approximately doubled the yield of DMPO-OH adduct, and higher Quin concentration increased the spin adduct signal even more. When DMPO-OH was pre-formed using Ti³⁺/hydrogen peroxide followed by peroxide removal with catalase, only addition of Quin-Fe(II), but not Fe(II), Fe(III), or Quin-Fe(III), significantly promoted decomposition of pre-formed DMPO-OH. Furthermore, reaction of Quin-Fe(II) with hydrogen peroxide leads to initial iron oxidation followed by appearance of iron redox cycling, detected as slow accumulation of ferrous ferrozine complex. This phenomenon cannot be abolished by subsequent addition of catalase. Thus, we propose that redox cycling of iron by a Quin derivative, formed by initial attack of hydroxyl radicals on Quin, rather than effects of iron complexes on DMPO-OH stability or redox cycling by hydrogen peroxide, is responsible for enhanced DMPO-OH signal in the presence of Quin. The present observations suggest that Quin-Fe(II) complexes display significant pro-oxidant characteristics that could have implications for Quin neurotoxicity.     

Evaluation of the association of mercury(II) with some dicysteinyl tripeptides

The present study was undertaken to gain insight into the associations of mercury(II) with dicysteinyl tripeptides in buffered media at pH 7.4. We investigated the effects of increasing the distance between cysteinyl residues on mercury(II) associations and complex formations. The peptide–mercury(II) formation constants and their associated thermodynamic parameters in 3-(N-morpholino)propanesulfonic acid (MOPS) buffered solutions were evaluated by isothermal titration calorimetry. Complexes formed in different relative ratios of mercury(II) to cysteinyl peptides in ammonium formate buffered solutions were characterized by LTQ Orbitrap mass spectrometry. The results from these studies show that n-alkyl dicysteinyl peptides (CP 1–4), and an aryl dicysteinyl peptide (CP 5) can serve as effective “double anchors” to accommodate the coordination sites of mercury(II) to form predominantly one-to-one Hg(peptide) complexes. The aryl dicysteinyl peptide (CP 5) also forms the two-to-two Hg₂(peptide)₂ complex. In the presence of excess peptide, Hg(peptide)₂ complexes are also detected. Notably, increasing the distance between the ligating groups or “anchor points” in CP 1–5 does not significantly affect their affinity for mercury(II). However, the enthalpy change (ΔH) values (ΔH₁ ∼ −91 kJ mol⁻¹ and ΔH₂ ∼ −66 kJ mol⁻¹) for complex formation between CP 4 and 5 with mercury(II) are about one and a half times larger than the related values for CP 1, 2 and 3 (ΔH₁ ∼ −66 kJ mol⁻¹ and ΔH₂ ∼ 46 kJ mol⁻¹). The corresponding entropy change (ΔS) values (ΔS₁ ∼ −129 J K⁻¹ mol⁻¹ and ΔS₂ ∼ −116 J K⁻¹ mol⁻¹) of the structurally larger dicysteinyl peptides CP 4 and 5 are less entropically favorable than for CP 1, 2 and 3 (ΔS₁ ∼ −48 J K⁻¹ mol⁻¹ and ΔS₂ ∼ −44 J K⁻¹ mol⁻¹). Generally, these associations result in a decrease in entropy, indicating that these peptide–mercury complexes potentially form highly ordered structures. The results from this study show that dicysteinyl tripeptides are effective in binding mercury(II) and they are promising motifs for the design of multi-cysteinyl peptides for binding more than one mercury(II) ion per peptide.     

Neocarzinostatin-based hybrid biocatalysts with a RNase like activity

A new zinc(II)-cofactor coupled to a testosterone anchor, zinc(II)-N,N-bis(2-pyridylmethyl)-1,3-diamino-propa-2-ol-N′(17′-succinimidyltestosterone) (Zn-Testo-BisPyPol) 1-Zn has been synthesized and fully characterized. It has been further associated with a neocarzinostatin variant, NCS-3.24, to generate a new artificial metalloenzyme following the so-called ‘Trojan horse’ strategy. This new 1-Zn-NCS-3.24 biocatalyst showed an interesting catalytic activity as it was found able to catalyze the hydrolysis of the RNA model HPNP with a good catalytic efficiency (k_cat/K_M = 13.6 M⁻¹ s⁻¹ at pH 7) that places it among the best artificial catalysts for this reaction. Molecular modeling studies showed that a synergy between the binding of the steroid moiety and that of the BisPyPol into the protein binding site can explain the experimental results, indicating a better affinity of 1-Zn for the NCS-3.24 variant than testosterone and testosterone-hemisuccinate themselves. They also show that the artificial cofactor entirely fills the cavity, the testosterone part of 1-Zn being bound to one the two subdomains of the protein providing with good complementarities whereas its metal ion remains widely exposed to the solvent which made it a valuable tool for the catalysis of hydrolysis reactions, such as that of HPNP. Some possible improvements in the ‘Trojan horse’ strategy for obtaining better catalysts of selective reactions will be further studied.     

[RuIII(medtra)(H2O)] (medtra = N-methylethylenediaminetriacetate) complex – A highly efficient NO inhibitor with low toxicity

Stopped-flow kinetic measurements were used to compare the reactivities of [Ru(medtra)(H₂O)] (medtra³⁻ = N-methylethylenediaminetriacetate) (1) and [Ru(hedtra)(H₂O)] (2) (hedtra³⁻ = N-hydroxyethylethylenediaminetriacetate) with NO in aqueous solution at 15 °C, pH 7.2 (phosphate buffer). The measured second-order rate constants (3 × 10³ and 6 × 10⁴ M⁻¹ s⁻¹ for 1 and 2, respectively) are three to four order of magnitudes lower than that for the reaction between [Ru^III(edta)(H₂O)]⁻ (3) with NO. However, NO scavenging studies of complexes 1–3, conducted by measuring the difference in nitrite production between treated and untreated murine macrophage cells, revealed that despite being less kinetically reactive toward NO, the [Ru(medtra)(H₂O)] complex exhibited the highest NO scavenging ability and lowest toxicity of compounds 1–3.     

One-electron oxidation and reduction of glycosaminoglycan chloramides: A kinetic study

Hypochlorous acid and its acid–base counterpart, hypochlorite ions, produced under inflammatory conditions, may produce chloramides of glycosaminoglycans, these being significant components of the extracellular matrix (ECM). This may occur through the binding of myeloperoxidase directly to the glycosaminoglycans. The N–Cl group in the chloramides is a potential selective target for both reducing and oxidizing radicals, leading possibly to more efficient and damaging fragmentation of these biopolymers relative to the parent glycosaminoglycans. In this study, the fast reaction techniques of pulse radiolysis and nanosecond laser flash photolysis have been used to generate both oxidizing and reducing radicals to react with the chloramides of hyaluronan (HACl) and heparin (HepCl). The strong reducing formate radicals and hydrated electrons were found to react rapidly with both HACl and HepCl with rate constants of 1–1.7×10⁸ and 0.7–1.2×10⁸ M⁻¹ s⁻¹ for formate radicals and 2.2×10⁹ and 7.2×10⁸ M⁻¹ s⁻¹ for hydrated electrons, respectively. The spectral characteristics of the products of these reactions were identical and were consistent with initial attack at the N–Cl groups, followed by elimination of chloride ions to produce nitrogen-centered radicals, which rearrange subsequently and rapidly to produce C-2 radicals on the glucosamine moiety, supporting an earlier EPR study by M.D. Rees et al. (J. Am. Chem. Soc. 125: 13719–13733; 2003). The oxidizing hydroxyl radicals also reacted rapidly with HACl and HepCl with rate constants of 2.2×10⁸ and 1.6×10⁸ M⁻¹ s⁻¹, with no evidence from these data for any degree of selective attack on the N–Cl group relative to the N–H groups and other sites of attack. The carbonate anion radicals were much slower with HACl and HepCl than hydroxyl radicals (1.0×10⁵ and 8.0×10⁴ M⁻¹ s⁻¹, respectively) but significantly faster than with the parent molecules (3.5×10⁴ and 5.0×10⁴ M⁻¹ s⁻¹, respectively). These findings suggest that these potential in vivo radicals may react in a site-specific manner with the N–Cl group in the glycosaminoglycan chloramides of the ECM, possibly to produce more efficient fragmentation. This is the first study therefore to conclusively demonstrate that reducing radicals react rapidly with glycosaminoglycan chloramides in a site-specific attack at the N–Cl group, probably to produce a 100% efficient biopolymer fragmentation process. Although less reactive, carbonate radicals, which may be produced in vivo via reactions of peroxynitrite with serum levels of carbon dioxide, also appear to react in a highly site-specific manner at the N–Cl group. It is not yet known if such site-specific attacks by this important in vivo species lead to a more efficient fragmentation of the biopolymers than would be expected for attack by the stronger oxidizing species, the hydroxyl radical. It is clear, however, that the N–Cl group formed under inflammatory conditions in the extracellular matrix does present a more likely target for both reactive oxygen species and reducing species than the N–H groups in the parent glycosaminoglycans.     

Preparation and spectroscopic properties of cobalt(III), nickel(II), copper(II) and zinc(II) complexes with a novel saturated macrocyclic ligand: 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22-docosahydrodibenzo-[b,i][1,4,8,11]tetraazacyclotetradecine

1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19, 20,21,22-Docosahydrodibenzo[b,i] [1,4,8,11] tetraazacyclotetradecine was prepared by hydrogenation of the benzo-analogue. Five isomers are feasible as a result of this hydrogenation but only two have been isolated: isomer A (melting point 158.5– 161.0 °C) and isomer B (melting point 194.5– 196.0 °C). The ¹³C NMR study was initiated to clear up the conformational differences between isomers. The cobalt(III), nickel(II), copper(II) and zinc(II) complexes of isomers A and B were prepared and investigated by near-ultraviolet, visible, infrared, NMR and ESR measurements. The ligand-field band in the 15 000-30 000 cm⁻¹ region for the cobalt(III), nickel(II) and copper(II) complexes provided information on their geometry around the central metal atom. That is to say, the cobalt(III) complexes are subjected to the octahedral ligand-field with axial elongation. The copper(II) complexes and the nickel- (II) complex of isomer A are subjected to the square- planar ligand-field in these complexes. The ligand- field bands for the nickel(II)complex of isomer B display the square-planar-distorted octahedral equilibrium in the coordinating solvent. ESR measurements for the copper(II) complexes also presented the spin Hamiltonian parameters in accord with the square- planar coordination. A strong band appearing at ca. 3200 cm⁻¹ was assigned to the N-H stretching mode and this band was slightly shifted to lower frequency upon metal coordination. The vibrational spectra and the conductance data provided evidences for the formation of the complexes with perchlorate ion as the counter ion. ¹³C NMR suggest that the complexes of isomer A are the cis-syn-cis form and the complexes of isomer B are the cis-anti-cis form.     

Synthesis,X-ray crystal structure,DNA/protein binding and cytotoxicity studies of five α-aminophosphonate N-derivatives

Five new α-aminophosphonates are synthesized and characterized by EA, FT-IR, ¹H NMR, ¹³C NMR, ³¹P NMR, ESI-MS and X-ray crystallography. The X-ray analyses reveal that the crystal structures of 1–5 are monoclinic or triclinic system with the space group P 21/c, P − 1, P − 1, P2(1)/c and P − 1, respectively. All P atoms of 1–5 have tetrahedral geometries involving two O-ethyl groups, one C_α atom, and a double bond O atom. The binding interaction of five new α-aminophosphonate N-derivatives (1–5) with calf thymus(CT)-DNA have been investigated by UV–visible and fluorescence emission spectrometry. The apparent binding constant (Kapp) values follows the order: 1 (3.38 × 10⁵ M⁻¹) > 2 (3.04 × 10⁵ M⁻¹) > 4 (2.52 × 10⁵ M⁻¹) > 5 (2.32 × 10⁵ M⁻¹) > 3 (2.10 × 10⁵ M⁻¹), suggesting moderate intercalative binding mode between the compounds and DNA. In addition, fluorescence spectrometry of bovine serum albumin (BSA) with the compounds 1–5 showed that the quenching mechanism might be a static quenching procedure. For the compounds 1–5, the number of binding sites were about one for BSA and the binding constants follow the order: 1 (2.72 × 10⁴ M⁻¹) > 2 (2.27 × 10⁴ M⁻¹) > 4 (2.08 × 10⁴ M⁻¹) > 5 (1.79 × 10⁴ M⁻¹) > 3 (1.17 × 10⁴ M⁻¹). Moreover, the DNA cleavage abilities of 1 exhibit remarkable changes and the in vitro cytotoxicity of 1 on tumor cells lines (MCF-7, HepG2 and HT29) have been examined by MTT and shown antitumor effect on the tested cells.     

Kinetics and mechanism of auto- and copper-catalyzed oxidation of 1,4-naphthohydroquinone

Although quinones represent a class of organic compounds that may exert toxic effects both in vitro and in vivo, the molecular mechanisms involved in quinone species toxicity are still largely unknown, especially in the presence of transition metals, which may both induce the transformation of the various quinone species and result in generation of harmful reactive oxygen species. In this study, the oxidation of 1,4-naphthohydroquinone (NH₂Q) in the absence and presence of nanomolar concentrations of Cu(II) in 10 mM NaCl solution over a pH range of 6.5–7.5 has been investigated, with detailed kinetic models developed to describe the predominant mechanisms operative in these systems. In the absence of copper, the apparent oxidation rate of NH₂Q increased with increasing pH and initial NH₂Q concentration, with concomitant oxygen consumption and peroxide generation. The doubly dissociated species, NQ²⁻, has been shown to be the reactive species with regard to the one-electron oxidation by O₂ and comproportionation with the quinone species, both generating the semiquinone radical (NSQ⁻). The oxidation of NSQ⁻ by O₂ is shown to be the most important pathway for superoxide (O₂⁻) generation with a high intrinsic rate constant of 1.0×10⁸ M⁻¹ s⁻¹. Both NSQ⁻ and O₂⁻ served as chain-propagating species in the autoxidation of NH₂Q. Cu(II) is capable of catalyzing the oxidation of NH₂Q in the presence of O₂ with the oxidation also accelerated by increasing the pH. Both the uncharged (NH₂Q⁰) and the mono-anionic (NHQ⁻) species were found to be the kinetically active forms, reducing Cu(II) with an intrinsic rate constant of 4.0×10⁴ and 1.2×10⁷ M⁻¹ s⁻¹, respectively. The presence of O₂ facilitated the catalytic role of Cu(II) by rapidly regenerating Cu(II) via continuous oxidation of Cu(I) and also by efficient removal of NSQ⁻ resulting in the generation of O₂⁻. The half-cell reduction potentials of various redox couples at neutral pH indicated good agreement between thermodynamic and kinetic considerations for various key reactions involved, further validating the proposed mechanisms involved in both the autoxidation and the copper-catalyzed oxidation of NH₂Q in circumneutral pH solutions.     

Hydrogen peroxide metabolism and sensing in human erythrocytes: A validated kinetic model and reappraisal of the role of peroxiredoxin II

Hydrogen peroxide (H₂O₂) metabolism in human erythrocytes has been thoroughly investigated, but unclear points persist. By integrating the available data into a mathematical model that accurately represents the current understanding and comparing computational predictions to observations we sought to (a) identify inconsistencies in present knowledge, (b) propose resolutions, and (c) examine their functional implications. The systematic confrontation of computational predictions with experimental observations of the responses of intact erythrocytes highlighted the following important discrepancy. The high rate constant (10⁷–10⁸ M⁻¹ s⁻¹) for H₂O₂ reduction determined for purified peroxiredoxin II (Prx2) and the high abundance of this protein indicate that under physiological conditions it consumes practically all the H₂O₂. However, this is inconsistent with extensive evidence that Prx2’s contribution to H₂O₂ elimination is comparable to that of catalase. Models modified such that Prx2’s effective peroxidase activity is just 10⁵ M⁻¹ s⁻¹ agree near quantitatively with extensive experimental observations. This low effective activity is probably due to a strong but readily reversible inhibition of Prx2’s peroxidatic activity in intact cells, implying that the main role of Prx2 in human erythrocytes is not to eliminate peroxide substrates. Simulations of the responses to physiological H₂O₂ stimuli highlight that a design combining abundant Prx2 with a low effective peroxidase activity spares NADPH while improving potential signaling properties of the Prx2/thioredoxin/thioredoxin reductase system.     

The palladium(II) promoted hydrolysis of the methyl esters of glycyl-L-leucine,glycyl-L-alanine and L-alanylglycine

The palladium(II)-promoted hydrolysis of the methyl esters of glycyl-L-leucine, glycyl-L-alanine and L-alanylglycine have been studied at 25 °C and I=0.1 M in the pH range 4–5. At a 1:1 metal to ligand ratio the peptide esters act as tridentate ligands, donation occurring via the terminal amino group, the deprotonated amide nitrogen, and the carbonyl group of the ester. Due to the high Lewis acidity of Pd(II) rapid hydrolysis of the ester function by water and hydroxide ion occurs. Rate constants k_OH and k_H2O have been obtained for base hydrolysis and water hydrolysis of the coordinated peptide esters at 25 °C. The rate constants for base hydrolysis are 3.4 X 10⁶ M⁻¹ s⁻¹ (L-alaglyOMe), 6.4 X 10⁶ M⁻¹ s⁻¹ (gly-L-alaOMe) and 2.3 X 10⁷ M⁻¹ s⁻¹ (gly-L-leuOMe). Base hydrolysis of the coordinated peptide esters is at least 10⁶ times that of the free unprotonated ligand. Activation parameters have been obtained for both water and base hydrolysis of the Pd(II) complex of methyl L-alanylglycinate and possible mechanisms for the hydrolyses are considered.     

Triazolopyridyl ketones as a novel class of antileishmanial agents. DNA binding and BSA interaction

A new series of triazolopyridyl pyridyl ketones has been synthetized by regioselective lithiation of the corresponding [1,2,3]triazolo[1,5-a]pyridine at 7 position followed by reaction with different electrophiles. The in vitro antileishmanial activity of these compounds was evaluated against Leishmania infantum, Leishmania braziliensis, Leishmania guyanensis and Leishmania amazonensis. Compounds 6 and 7 were found to be the most active leishmanicidal agents. Both of them showed activities at micromolar concentration against cultured promastigotes of Leishmania spp. (IC₅₀ = 99.8–26.8 μM), without cytotoxicity on J774 macrophage cells. These two compounds were also tested in vivo in a murine model of acute infection by L. infantum. The triazolopyridine derivative 6 was effective against both spleen and liver parasites forms, while 7 was inactive against liver parasites. Mechanistic aspects of the antileishmanial activity were investigated by means of DNA binding studies (UV-titration and viscosimetry). Results have revealed that these active ligands are able to interact strongly with DNA [K_b = 1.14 × 10⁵ M⁻¹ (6) and 3.26 × 10⁵ M⁻¹ (7)]. Moreover, a DNA groove binding has been proposed for both 6 and 7. To provide more insight on the mode of action of compounds 6 and 7 under biological conditions, their interaction with bovine serum albumin (BSA) was monitored by fluorescence titrations and UV–visible spectroscopy. The quenching constants and binding parameters were determined. Triazolopyridine ketones 6 and 7 have exhibited significant affinity towards BSA [K_b = 2.5 × 10⁴ M⁻¹ (6) and 1.9 × 10⁴ M⁻¹ (7)]. Finally, to identify the binding location of compounds 6 and 7 on the BSA, competitive binding experiments were carried out, using warfarin, a characteristic marker for site I, and ibuprofen as one for site II. Results derived from these studies have indicated that both compounds interact at BSA site I and, to a lesser extent, at site II.     

The synthesis and structural properties of [M(dippe)(η2-C4H4S)] complexes of Pd and Pt and comparison with their Ni analog

X-ray structural and NMR spectroscopic data for the ring-opened thiophene complexes [Pd(dippe)(T)] (2), and [Pt(dippe)(T)] (3) are now presented. The complex [Ni(dippe)(T)] (1), where T = (η²-C,S-C₄H₄S), was reported by our group, previously.The structural and bonding properties of complexes 2 and 3 were compared with those of complex 1. DFT calculations were carried out to rationalize their relative stabilities and structural properties. Compound 1 loses thiophene at ambient temperature in solution, while compound 2 decomposes rapidly in both acetone-d₆ and THF-d₈ with k_obs = 7.15(9) × 10⁻⁵ and 7.7(3) × 10⁻⁵ s⁻¹, respectively, to give products that varied by solvent. Complex 3 does not lose thiophene at temperatures below 100 °C. The ΔG⁰ values determined from DFT calculations are consistent with the observed stabilities of the complexes. The single crystal X-ray structures of all three complexes contain a disordered thienyl fragment in the asymmetric unit due to the interchange of the position of sulfur in the metal-inserted thiophenic ring. The thiophenic moiety is relatively flat in 1, 2 and 3, which is attributed to the open ligand environment at the M(dippe) fragment. All three complexes possess square-planar geometry around the metal center and have bond-length alternation among the thiophenic carbons, which indicates double bond localization. The calculated bond lengths are in good agreement with experimental data. Molecular orbital (MO) and natural bonding orbital (NBO) analyses were carried out to rationalize the results.     

One-step partial synthesis of (±)-asperteretone B and related hPTP1B1–400 inhibitors from butyrolactone I

Protein tyrosine phosphatase 1B (PTP1B) is a validated target for developing antiobesity, antidiabetic and anticancer drugs. Over the past years, several inhibitors of PTP1B have been discovered; however, none has been approved by the drug regulatory agencies. Interestingly, the research programs focused on discovering PTP1B inhibitors typically use truncated structures of the protein (PTP1B_1-300, 1–300 amino acids), leading to the loss of valuable information about the inhibition and selectivity of ligands and repeatedly misleading the optimization of putative drug leads. Up to date, only six inhibitors of the full-length protein (hPTP1B_1-400), with affinity constants ranging from 1.3 × 10⁴ to 3.3 × 10⁶ M⁻¹, have been reported. Towards the discovery of new ligands of the full-length human PTP1B (hPTP1B_1-400) from natural sources, herein we describe the isolation of a γ-lactone (1, butyrolactone I) from the fungus Aspergillus terreus, as well as the semisynthesis, inhibitory properties (in vitro and in silico), and the structure-activity relationship of a set of butyrolactone derivatives (1 and 2, and 6–12) as hPTP1B_1-400 inhibitors, as well as the affinity constant (k_a = 2.2 × 10⁵ M⁻¹) of the 1-hPTP1B₁–₄₀₀ complex, which was determined by fluorescence quenching experiments, after the inner filter effect correction.     

Arguments against the significance of the Fenton reaction contributing to signal pathways under in vivo conditions

One of the common explanations for oxidative stress in the physiological milieu is based on the Fenton reaction, i.e. the assumption that radical chain reactions are initiated by metal-catalyzed electron transfer to hydrogen peroxide yielding hydroxyl radicals. On the other hand — especially in the context of so-called “iron switches” — it is postulated that cellular signaling pathways originate from the interaction of reduced iron with hydrogen peroxide.

Using fluorescence detection and EPR for identification of radical intermediates, we determined the rate of iron complexation by physiological buffer together with the reaction rate of concomitant hydroxylations of aromatic compounds under aerobic and anaerobic conditions. With the obtained overall reaction rate of 1,700 M^-1s^-1 for the buffer-dependent reactions and the known rates for Fenton reactions, we derive estimates for the relative reaction probabilities of both processes.

As a consequence we suggest that under in vivo conditions initiation of chain reactions by hydroxyl radicals generated by the Fenton reaction is of minor importance and hence metal-dependent oxidative stress must be rather independent of the so-called “peroxide tone”. Furthermore, it is proposed that — in the low (subtoxic) concentration range — hydroxylated compounds derived from reactions of “non-free” (crypto) OH radicals are better candidates for iron-dependent sensing of redox-states and for explaining the origin of cellular signals than the generation of “free” hydroxyl radicals.