A Synthetic Model for the Inhibition of Glutathione Peroxidase by Antiarthritic Gold Compounds

In this paper, inhibition of the glutathione peroxidase activity of two synthetic organoselenium compounds, bis[2-(N,N-dimethylamino)benzyl]diselenide (5) and bis[2-(N,N-dimethylamino)benzyl]selenide (9), by gold(I) thioglucose (1), chloro(triethylphosphine)gold(I), chloro(trimethylphosphine)gold(I), and chloro(triphenylphosphine)gold(I) is described. The inhibition is found to be competitive with respect to a peroxide (H2O2) substrate and noncompetitive with respect to a thiol (PhSH) cosubstrate. The diselenide 5 reacts with PhSH to produce the corresponding selenol (6), which upon treatment with 1 equiv of gold(I) chlorides produces the corresponding gold selenolate complexes 11−13. However, the addition of 1 equiv of selenol 6 to complexes 11−13 leads to the formation of bis-selenolate complex 14 by ligand displacement reactions involving the elimination of phosphine ligands. The phosphine ligands eliminated from these reactions are further converted to the corresponding phosphine oxides (R3PO) and selenides (R3PSe). In addition to the replacement of the phosphine ligand by selenol 6, an interchange between two different phosphine ligands is also observed. For example, the reaction of complex 11 having a trimethylphosphine ligand with triphenylphosphine produces complex 13 by phosphine interchange reactions via the formation of intermediates 15 and 16. The reactivity of selenol 6 toward gold(I) phosphines is found to be similar to that of selenocysteine.

Inhibition of Peroxidase-Catalyzed Iodination by Cephalosporins: Metallo-beta-Lactamase-Induced Antithyroid Activity of Antibiotics

Broad-spectrum antibiotics with heterocyclic side chains strongly inhibit peroxidase-catalyzed iodination in the presence of metallo--lactamase. This suggests that antibiotic resistance due to hydrolysis of the -lactam ring in antibiotics would have negative effects on thyroid activity.

Amide-Based Glutathione Peroxidase Mimics: Effect of Secondary and Tertiary Amide Substituents on Antioxidant Activity

A series of secondary and tertiary amide-substituted diselenides were synthesized and studied for their GPx-like antioxidant activities using H2O2 Cum-OOH, and tBuOOH as substrates and PhSH as thiol co-substrate.The effect of substitution at the free -NH group of the amide moiety in the sec-amide-based diselenides on GPx activity was analyzed by detailed experimental and theoretical methods. It is observed that substitution at the free -NH group significantly enhances the GPx-like activities of the sec-amide-based diselenides, mainly by reducing the Se center dot center dot center dot O nonbonded interactions. The reduction in strength of the Se center dot center dot center dot O interaction upon introduction of N,N-dialkyl substituents not only prevents the undesired thiol exchange reactions, but also reduces the stability of selenenyl sulfide intermediates. This leads to a facile disproportionation of the selenenyl sulfide to the corresponding diselenide, which enhances the catalytic activity. The mechanistic investigations indicate that the reactivity of diselenides having sec-or tert-amide moieties with PhSH is extremely slow; indicating that the first step of the catalytic cycle involves the reaction between the diselenides and peroxide to produce the corresponding selenenic and seleninic acids.

Oxidase-peroxidase enzymes of Datura innoxia. Oxidation of formylphenylacetic acid ethyl ester

An enzyme system from Datura innoxia roots oxidizing formylphenylacetic acid ethyl ester was purified 38-fold by conventional methods such as (NH4)2SO4 fractionation, negative adsorption on alumina Cy gel and chromatography on DEAE-cellulose. The purified enzyme was shown to catalyse the stoicheiometric oxidation of formylphenylacetic acid ethyl ester to benzoylformic acid ethyl ester and formic acid, utilizing molecular O2. Substrate analogues such as phenylacetaldehyde and phenylpyruvate were oxidized at a very low rate, and formylphenylacetonitrile was an inhilating agents, cyanide, thiol compounds and ascorbic acid. This enzyme was identical with an oxidase-peroxidase isoenzyme. Another oxidase-peroxidase isoenzyme which separated on DEAE-chromatography also showed formylphenylacetic acid ethyl ester oxidase activity, albeit to a lesser extent. The properties of the two isoenzymes of the oxidase were compared and shown to differ in their oxidation and peroxidation properties. The oxidation of formylphenylacetic acid ethyl ester was also catalysed by horseradish peroxidase. The Datura isoenzymes exhibited typical haemoprotein spectra. The oxidation of formylphenylacetic acid ethyl ester was different from other peroxidase-catalysed reactions in not being activated by either Mn2+ or monophenols. The oxidation was inhibited by several mono- and poly-phenols and by catalase. A reaction mechanism for the oxidation is proposed.

Oxidase-peroxidase enzymes of Datura innoxia. Oxidation of reduced nicotinamide-adenine dinucleotide in the presence of formylphenylacetic acid ethyl ester

The oxidase-peroxidase from Datura innoxia which catalyses the oxidation of formylphenylacetic acid ethyl ester to benzoylformic acid ethyl ester and formic acid was also found to catalyse the oxidation of NADH in the presence of Mn2+ and formylphenylacetic acid ethyl ester. NADH was not oxidized in the absence of formylphenylacetic acid ethyl ester, although formylphenylacetonitrile or phenylacetaldehyde could replace it in the reaction. The reaction appeared to be complex and for every mol of NADH oxidized 3-4 g-atoms of oxygen were utilized, with a concomitant formation of approx. 0.8 mol of H2O2, the latter being identified by the starch-iodide test and decomposition by catalase. Benzoylformic acid ethyl ester was also formed in the reaction, but in a nonlinear fashion, indicating a lag phase. In the absence of Mn2+, NADH oxidation was not only very low, but itself inhibited the formation of benzoylformic acid ethyl ester from formylphenylacetic acid ethyl ester. A reaction mechanism for the oxidation of NADH in the presence of formylphenylacetic acid ethyl ester is proposed.

The purification and properties of peroxidase in Mycobacterium tuberculosis H37Rv and its possible role in the mechanism of action of isonicotinic acid hydrazide

Peroxidase from Mycobacterium tuberculosis H37Rv was purified to homogeneity. The homogeneous protein exhibits catalase and Y (Youatt's)-enzyme activities in addition to peroxidase activity. Further confirmation that the three activities are due to a single enzyme was accomplished by other criteria, such as differential thermal inactivation, sensitivity to different inhibitors, and co-purification. The Y enzyme (peroxidase) was separated from NADase (NAD+ glycohydrolase) inhibitor by gel filtration on Sephadex G-200. The molecular weights of peroxidase and NADase inhibitor, as determined by gel filtration, are 240000 and 98000 respectively. The Y enzyme shows two Km values for both isoniazid (isonicotinic acid hydrazide) and NAD at low and high concentrations. Analysis of the data by Hill plots revealed that the enzyme has one binding site at lower substrate concentrations and more than one at higher substrate concentration. The enzyme contains 6g-atoms of iron/mol. Highly purified preparations of peroxidases from different sources catalyse the Y-enzyme reaction, suggesting that the nature of the reaction may be a peroxidatic oxidation of isoniazid. Moreover, the Y-enzyme reaction is enhanced by O2. Isoniazid-resistant mutants do not exhibit Y-enzyme, peroxidase or catalase activities, and do not take up isoniazid. The Y-enzyme reaction is therefore implicated in the uptake of the drug.

Synthesis and Structure-Activity Correlation Studies of Secondary- and Tertiary-Amine-Based Glutathione Peroxidase Mimics

In this study, a series of seeondary- and tertiary-amino-substituted diaryl diselenides were synthesized and studied for their glutathione peroxidase (GPx) like antioxidant activities with H2O2, cumene hydroperoxide, or tBuOOH as substrates and with PhSH or glutathione (GSH) as thiol cosubstrates. This study reveals that replacement of the tert-amino groups in benzylamine-based diselenides by sec-amino moieties drastically enhances the catalytic activities in both the aromatic thiol (PhSH) and GSH assay systems. Particularly, the N-propyl- and N-isopropylamino-substituted diselenides are 8-18 times more active than the corresponding N,N-dipropyl- and N,N-diisopropylamine-based compounds in all three peroxide systems when GSH is used as the thiol cosubstrate. Although the catalytic mechanism of sec-amino-substituted disclenides is similar to that of the tert-amine-based compounds, differences in the stability and reactivity of some of the key intermediates account for the differences in the GPx-like activities. it is observed that the sec-amino groups are better than the tert-amino moieties for generating the catalytically active selenols. This is due to the absence of any significant thiol-exchange reactions in the selenenyl sulfides derived from sec-amine-based diselenides. Furthermore, the seleninic acids (RSeO2H) derived from the sec-amine-based compounds are more stable toward further reactions with peroxides than their tert-amine-based analogues.

Synthesis, Structure, Spirocyclization Mechanism, and Glutathione Peroxidase-like Antioxidant Activity of Stable Spirodiazaselenurane and Spirodiazatellurane

The first examples of stable spirodiazaselenurane and spirodiazatellurane were synthesized by oxidative spirocyclization of the corresponding diaryl selenide and telluride and were structurally characterized. X-ray crystal structures of the spirodiazaselenurane and spirodiazatellurane suggest that the structures are distorted trigonal bipyramidal (TBP) with the electronegative nitrogen atoms occupying the apical positions and two carbon atoms and the lone pair of Se/Te occupying the equatorial positions. Interestingly, the spirodiazatellurane underwent spontaneous chiral resolution during crystallization, and the absolute configurations of its enantiomers were confirmed by single-crystal X-ray analyses. A detailed mechanistic study indicates that the cyclization to spirodiazaselenurane and spirodiazatellurane occurs via selenoxide and telluroxide intermediates. The chalcogenoxides cyclize to the corresponding spiro compounds in a stepwise manner via the involvement of hydroxyl chalcogenurane intermediates, and the activation energy for them spirocyclization reaction decreases in the order S > Se > Te. In addition to the synthesis, characterization, and mechanism of cyclization, the glutathione peroxidase (GPx) mimetic activity of the newly synthesized compounds was evaluated. These studies suggest that the tellurium compounds are more effective as GPx mimics than their selenium counterparts due to the fast oxidation of the tellurium center in the presence of peroxide and the involvement of an efficient redox cycle between the telluride and telluroxide intermediate.

Inhibition of peroxidase-catalyzed protein tyrosine nitration by antithyroid drugs and their analogues

In this paper, we describe the effect of some commonly used thiourea-based antithyroid drugs and their analogues on the peroxidase-catalyzed nitration reactions. The nitration of bovine serum albumin (BSA) and cytochrome c was studied using the antibody against 3-nitro-L-tyrosine. This study reveals that the thione-based antithyroid drugs effectively inhibit lactoperoxidase (LPO)-catalyzed nitration of BSA. These compounds show very weak inhibition towards the nitration of cytochrome c. Some of these compounds also inhibit myeloperoxidase (MPO)-catalyzed nitration of L-tyrosine. A structure-activity correlation study on the peroxidase-catalyzed nitration of L-tyrosine reveals that the presence of thione/selone moiety is important for the inhibition. Although the presence of a free N-H group adjacent to C=S moiety is necessary for most of the thiones to inhibit the LPO-catalyzed nitration, the corresponding selones do not require the presence of any free N-H group for their activity. Furthermore, experiments with different concentrations of H2O2 suggest that the antithyroid drugs and related thiones inhibit the nitration reaction mainly by coordinating to the Fe(III)-center of the enzyme active site as previously proposed for the inhibition of peroxidase-catalyzed iodination. On the other hand, the selenium compounds inhibit the nitration by scavenging H2O2 without interacting with the enzyme active site. This assumption is based on the observations that catalase effectively inhibits tyrosine nitration by scavenging H2O2, which is one of the substrates for the nitration. In contrast, superoxide dismutase (SOD) does not alter the nitration reactions, indicating the absence of superoxide radical anion (O-2 center dot(-)) during the peroxidase-catalyzed nitration reactions. (C) 2010 Elsevier B.V. All rights reserved.

Functional Mimics of Glutathione Peroxidase: Bioinspired Synthetic Antioxidants

Oxidative stress is caused by an imbalance between the production of reactive oxygen species (ROS) and the biological system's ability to detoxify these reactive intermediates. Mammalian cells have elaborate antioxidant defense mechanisms to control the damaging effects of ROS. Glutathione peroxidase (GPx), a selenoenzyme, plays a key role in protecting the organism from oxidative damage by catalyzing the reduction of harmful hydroperoxides with thiol a ``catalytic triad'' with tryptophan and glutamine, which cofactors. The selenocysteine residue at the active site forms activates the selenium moiety for an efficient reduction of peroxides. After the discovery that ebselen, a synthetic organoselenium compound, mimics the catalytic activity of GPx both in vitro and in vivo, several research groups developed a number of small-molecule selenium compounds as functional mimics of GPx, either by modifying the basic structure of ebselen or by incorporating some structural features of the native enzyme. The synthetic mimics reported in the literature can be classified in three major categories: (i) cyclic selenenyl amides having a Se-N bond, (ii) diaryl diselenides, and (iii) aromatic or aliphatic monoselenides. Recent studies show that ebselen exhibits very poor GPx activity when aryl or benzylic thiols such as PhSH or BnSH are used as cosubstrates. Because the catalytic activity of each GPx mimic largely depends on the thiol cosubstrates used, the difference in the thiols causes the discrepancies observed in different studies. In this Account, we demonstrate the effect of amide and amine substituents on the GPx activity of various organoselenium compounds. The existence of strong Se ... O/N interactions in the selenenyl sulfide intermediates significantly reduces the GPx activity. These interactions facilitate an attack of thiol at selenium rather than at sulfur, leading to thiol exchange reactions that hamper the formation of catalytically active selenol. Therefore, any substituent capable of enhancing the nucleophilic attack of thiol at sulfur in the selenenyl sulfide state would enhance the antioxidant potency of organoselenium compounds. Interestingly, replacement of the sec-amide substituent by a tert-amide group leads to a weakening of Se ... 0 interactions in the selenenyl sulfide intermediates. This modification results in 10- to 20-fold enhancements in the catalytic activities. Another strategy involving the replacement of tert-amide moieties by tert-amino substituents further increases the activity by 3- to 4-fold. The most effective modification so far in benzylamine-based GPx mimics appears to be either the replacement of a tert-amino substituent by a sec-amino group or the introduction of an additional 6-methoxy group in the phenyl ring. These strategies can contribute to a remarkable enhancement in the GPx activity. In addition to enhancing catalytic activity, a change in the substituents near the selenium moiety alters the catalytic mechanisms. The mechanistic investigations of functional mimics are useful not only for understanding the complex chemistry at the active site of GPx but also for designing and synthesizing novel antioxidants and anti-inflammatory agents.

Competing peroxidase and oxidase reactions in scopoletin-dependent H2O2-initiated oxidation of NADH by horseradish peroxidase

Addition of NADH inhibited the peroxidative loss of scopoletin in presence of horseradish peroxidase and H2O2 and decreased the ratio of scopoletin (consumed):H2O2 (added). Concomitantly NADH was oxidized and oxygen was consumed with a stoichiometry of NADH: O-2 of 2:1. On step-wise addition of a small concentration of H2O2 a high rate of NADH oxidation was obtained for a progressively decreasing time period followed by termination of the reaction with NADH:H2O2 ratio decreasing from about 40 to 10. The rate of NADH oxidation increased linearly with increase in scopoletin concentration. Other phenolic compounds including p-coumarate also supported this reaction to a variable degree. A 418-nm absorbing compound;d accumulated during oxidation of NADH. The effectiveness of a small concentration of H2O2 in supporting NADH oxidation increased in presence of SOD and decreased in presence of cytochrome c, but the reaction terminated even in their presence. The results indicate that the peroxidase is not continuously generating H2O2 during scopolerin-mediated NADH oxidation and that both peroxidase and oxidase reactions occur simultaneously competing for an active form of the enzyme.

Total peroxidase activity and peroxidase isoforms as modified by salt stress in two cultivars of fox-tail millet with differential salt tolerance

The effect of NaCl on total peroxidase activity, induction of isoperoxidases and lipid peroxidation in 5-day-old seedlings of two contrasting genotypes of Setaria italica L. (Prasad, a salt tolerant cultivar and Lepakshi, a salt susceptible cultivar), was studied. Total peroxidase activity increased under NaCl salinity and the degree of elevation in the activity was salt concentration dependent. Nevertheless, a greater activity was recorded in the tolerant cultivar (cv Prasad) compared to the susceptible (cv Lepakshi) one in all days of sampling. Further, the pattern of isoperoxidases was modified during stress conditions as evident from the electrophoregrams. Although, five acidic isoforms were detected in both cultivars, differences were found between the cultivars. Furthermore, it was observed that acidic isoperoxidases were strongly expressed and an acidic isoperoxidase, A(3p) (27 kDa) is specifically found in the tolerant cultivar (cv Prasad) under NaCl stress. This isoform was partially purified and found to be thermostable with pr 5.5 and the optimum pH 7.4. A close correlation exists between the rate of lipid peroxidation in terms of malonaldehyde (MDA) content and total peroxidase activity per gram fresh weight with salt tolerance of the two cultivars. The tolerant cultivar (cv Prasad) had low MDA content and high total peroxidase activity than the susceptible variety (cv Lepakshi) during salinity stress. (C) 1999 Published by Elsevier Science Ireland Ltd. All rights reserved.

Inhibition of peroxynitrite- and peroxidase-mediated protein tyrosine nitration by imidazole-based thiourea and selenourea derivatives

In the present study, the synthesis and characterization of a series of N-methylimidazole-based thiourea and selenourea derivatives are described. The new compounds were also studied for their ability to inhibit peroxynitrite (PN)- and peroxidase-mediated nitration of protein tyrosine residues. It has been observed that the selenourea derivatives are more efficient than the thiourea-based compounds in the inhibition of protein nitration. The higher activity of selenoureas as compared to that of the corresponding thioureas can be ascribed to the zwitterionic nature of the selenourea moiety. Single crystal X-ray diffraction studies on some of the thiourea and selenourea derivatives reveal that the C S bonds in thioureas possess more of double bond character than the C=Se bonds in the corresponding selenoureas. Therefore, the selenium compounds can react with PN or hydrogen peroxide much faster than their sulfur analogues. The reactions of thiourea and selenourea derivatives with PN or hydrogen peroxide produce the corresponding sulfinic or seleninic acid derivatives, which upon elimination of sulfurous/selenous acids produce the corresponding N-methylimdazole derivatives.

Growth of Polypyrrole-Horseradish Peroxidase Microstructures for H2O2 Biosensor

Conducting polymer microstructures for enzymatic biosensors are developed by a facile electrochemical route. Horseradish peroxide (HRP)-entrapped polypyrrole (PPy) films with bowl-shaped microstructures are developed on stainless steel (SS 304) substrates by a single-step process. Potentiodynamic scanning/cyclic voltammetry is used for generation of PPy microstructures using electrogenerated oxygen bubbles stabilized by zwitterionic surfactant/buffer N-2-hydroxyethylpiperazine N-2-ethanesulfonic acid as soft templates. Scanning electron microscopic images reveal the bowl-shaped structures surrounded by cauliflower-like fractal PPy films and globular nanostructures. Raman spectroscopy reveals the oxidized nature of the film. Sensing properties of PPy-HRP films for hydrogen peroxide (H2O2) are demonstrated. Electrochemical characterization of the sensor films is done by linear sweep voltammetry (LSV) and amperometry. LSV results indicated the reduction of H2O2 and linearity in response of the sensing film. The amperometric biosensor has a performance comparable to those in the literature with advantages of hard-template free synthesis procedure and a satisfactory sensitivity value of 12.8 mu A/(cm(2) . mM) in the range of 1-10 mM H2O2.

Tertiary amine-based glutathione peroxidase mimics: some insights into the role of steric and electronic effects on antioxidant activity

In this work, several tertiary amine-based diaryl diselenides were synthesized and evaluated for their glutathione peroxidase (GPx)-like antioxidant activities using hydrogen peroxide, tert-butyl hydroperoxide and cumene hydroperoxide as substrates and thiophenol (PhSH) and glutathione (GSH) as co-substrates. A comparison of the GPx-like activity of 4-methoxy-substituted N,N-dialkylbenzylamine-based diselenides with that of the corresponding 6-methoxy-substituted compounds indicates that the activity highly depends on the position of the methoxy substituent. Although the methoxy group at 4- and 6-position alters the electronic properties of selenium, the substitution at the 6-position provides the required steric protection for some of the key intermediates in the catalytic cycle. A detailed experimental and theoretical investigation reveals that the 6-methoxy substituent prevents the undesired thiol exchange reactions at the selenium centers in the selenenyl sulfide intermediates. The 6-methoxy substituent also prevents the formation of seleninic and selenonic acids. When PhSH is used as the thiol co-substrate, the 4-methoxy-substituted diselenides exhibit GPx-like activity similar to that of the parent compounds as the 4-methoxy substituent does not block the selenium center in the selenenyl sulfide intermediates from thiol exchange reactions. In contrast, the 4-methoxy substituent significantly enhances the GPx-like activity of the diselenides when glutathione (GSH) is used as the co-substrate. (C) 2012 Elsevier Ltd. All rights reserved.