Papers by Zilvinas Anusevičius
Archives of Biochemistry and Biophysics, 2001
Enterobacter cloacae NAD(P)H:nitroreductase (NR; EC 1.6.99.7) catalyzes the reduction of a series... more Enterobacter cloacae NAD(P)H:nitroreductase (NR; EC 1.6.99.7) catalyzes the reduction of a series of nitroaromatic compounds with steady-state bimolecular rate constants (k cat /K m ) ranging from 10 4 to 10 7 M ؊1 s ؊1 . In agreement with a previously proposed scheme of two-step four-electron reduction of nitroaromatics by NR (Koder, R. L., and Miller, A.-F. (1998) Biochim. Biophys. Acta 1387, 395-405), 2 mol NADH per mole mononitrocompound were oxidized. An oxidation of excess NADH by polinitrobenzenes, including explosives 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-Nmethylnitramine (tetryl), has been observed as a slower secondary process, accompanied by O 2 consumption. This type of "redox cycling" was not related to reactions of nitroaromatic anion-radicals, but was caused by the autoxidation of relatively stable reaction products. The initial reduction of tetryl and other polinitrophenyl-N-nitramines by E. cloacae NR was analogous to a two-step four-electron reduction mechanism of TNT and other nitroaromatics. The logs k cat /K m of all the compounds examined exhibited parabolic dependence on their enthalpies of single-electron or two-electron (hydride) reduction, obtained by quantum mechanical calculations. This type of quantitative structure-activity relationship shows that the reactivity of nitroaromatics towards E. cloacae nitroreductase depends mainly on their hydride accepting properties, but not on their particular structure, and does not exclude the possibility of multistep hydride transfer.
![Research paper thumbnail of Benzofuroxan (Benzo[1,2-c]1,2,5-oxadiazole N-oxide) Derivatives as Potential Energetic Materials: Studies on Their Synthesis and Properties](https://a.academia-assets.com/images/blank-paper.jpg)
Central European Journal of Energetic Materials, 2012
The objective of this work was to prepare benzofuroxan derivatives as new, dense, potentially ene... more The objective of this work was to prepare benzofuroxan derivatives as new, dense, potentially energetic materials and to investigate their properties, with the main focus being on 5,6-dinitrobenzofuroxan (5,6-DNBF). 5,6-DNBF was prepared by a 3-step reaction sequence: a) 1-azido-3-nitrobenzene was synthesized by diazotation of 3-nitroaniline with sodium nitrite and subsequent reaction with sodium azide in acetic/sulfuric mixed acids; b) it was nitrated with HNO3/H2SO4 to 1-azido-2,4,5-trinitrobenzene; c) thermal cyclization of the latter compound in a polar solvent gave the desired 5,6-DNBF (m.p. 177 °C). It was fully characterized by UV/VIS, FT-IR and NMR spectroscopy, mass spectrometry and single crystal X-ray diffraction. The density of the compound (X-ray) was found to be comparatively high (1.88 g/cm3), and to be superior to the previously known, isomeric energetic material − 4,6-dinitrobenzofuroxan (4,6-DNBF) (1.76 g/cm3). Furthermore, the synthesis of some other benzofuroxan derivatives, potentially interesting as high energy, density materials (HEDMs), has been carried out. The densities of the compounds obtained were calculated using ACD Labs software (version 4.0). Based on the results obtained, it could be concluded that 5,6-DNBF is one of the densest nitro derivatives of the benzofuroxan series, comparable to CL-14, CL-17, CL-18, and thus could have potential applications as an HEDM.
Biochimica Et Biophysica Acta - General Subjects, Sep 1, 2001
The mechanisms of cytotoxicity of polynitroaromatic explosives, an important group of environment... more The mechanisms of cytotoxicity of polynitroaromatic explosives, an important group of environmental pollutants, remain insufficiently studied so far. We have found that the rate constants of single-electron enzymatic reduction, and the enthalpies of single-electron reduction of nitroaromatic compounds (ΔHf(ArNO2−⋅)), obtained by quantum mechanical calculation, may serve as useful tools for the analysis of cytotoxicity of nitroaromatic explosives with respect to
Journal of Photochemistry and Photobiology B: Biology
![Research paper thumbnail of Article The Study of NADPH-Dependent Flavoenzyme-Catalyzed Reduction of Benzo[1,2-c]1,2,5-oxadiazole N-Oxides (Benzofuroxans)](https://attachments.academia-assets.com/117801442/thumbnails/1.jpg)
Abstract: The enzymatic reactivity of a series of benzo[1,2-c]1,2,5-oxadiazole N-oxides (benzofur... more Abstract: The enzymatic reactivity of a series of benzo[1,2-c]1,2,5-oxadiazole N-oxides (benzofuroxans; BFXs) towards mammalian single-electron transferring NADPH:cytochrome P-450 reductase (P-450R) and two-electron (hydride) transferring NAD(P)H:quinone oxidoreductase (NQO1) was examined in this work. Since the =N+ (→O)O − moiety of furoxan fragments of BFXs bears some similarity to the aromatic nitro-group, the reactivity of BFXs was compared to that of nitro-aromatic compounds (NACs) whose reduction mechanisms by these and other related flavoenzymes have been extensively investigated. The reduction of BFXs by both P-450R and NQO1 was accompanied by O2 uptake, which was much lower than the NADPH oxidation rate; except for annelated BFXs, whose reduction was followed by the production of peroxide. In order to analyze the possible quantitative structure-activity relationships (QSARs) of the enzymatic reactivity of the compounds, their electron-accepting potency and other reactivity ...
Chemija, 2020
Trypanothione reductase (TR) and trypanothione synthase (TS) are critical for the maintenance of ... more Trypanothione reductase (TR) and trypanothione synthase (TS) are critical for the maintenance of thiol-redox homeostasis and antioxidant protection in trypanosomal parasites, which cause African sleeping sickness and Chagas disease. Both enzymes are absent in mammals. Thus, the design of efficient and specific TR and TS inhibitors represents one of the pathways for a development of new antitrypanosomal drugs. 5-Vinylquinoline-substituted nitrofurans (n = 7), studied in this work, acted as un- or noncompetitive to trypanothione inhibitors of Trypanosoma congolense TR. Their inhibition constants (Ki) varied from 2.3 µM to 150 µM. We for the first time observed a parallelism between their antitrypanosomal in vitro activity and their efficacy as TR inhibitors. The inhibition of TS appears not to be a significant factor of trypanocidal activity of examined compounds.
Acta Biochimica Polonica, 2006
We aimed to elucidate the role of electronic and structural parameters of nitroaromatic compounds... more We aimed to elucidate the role of electronic and structural parameters of nitroaromatic compounds in their two-electron reduction by NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2). The multiparameter regression analysis shows that the reactivity of nitroaromatic compounds (n=38) increases with an increase in their single-electron reduction potential and the torsion angle between nitrogroup(s) and the aromatic ring. The binding efficiency of nitroaromatics in the active center of NQO1 exerted a less evident role in their reactivity. The reduction of nitroaromatics is characterized by more positive entropies of activation than the reduction of quinones. This points to a less efficient electronic coupling of nitroaromatics with the reduced isoalloxazine ring of FAD, and may explain their lower reactivity as compared to quinones. Another important but poorly understood factor enhancing the reactivity of nitroaromatics is their ability to bind at the dicumarol/quinone ...
Free radical biology & medicine, Jan 21, 2018
In microorganisms, flavohemoglobins (FHbs) containing FAD and heme (Fe, metHb) convert NO. into n... more In microorganisms, flavohemoglobins (FHbs) containing FAD and heme (Fe, metHb) convert NO. into nitrate at the expense of NADH and O. FHbs contribute to bacterial resistance to nitrosative stress. Therefore, inhibition of FHbs functions may decrease the pathogen virulence. We report here a kinetic study of the reduction of quinones and nitroaromatic compounds by S. aureus FHb. We show that this enzyme rapidly reduces quinones and nitroaromatic compounds in a mixed single- and two-electron pathway. The reactivity of nitroaromatics increased upon an increase in their single-electron reduction potential (E), whereas the reactivity of quinones poorly depended on their E with a strong preference for a 2-hydroxy-1,4-naphthoquinone structure. The reaction followed a 'ping-pong' mechanism. In general, the maximal reaction rates were found lower than the maximal presteady-state rate of FAD reduction by NADH and/or of oxyhemoglobin (HbFeO) formation (~130 s, pH 7.0, 25 °C), indicating...
Central European Journal of Energetic Materials, 2012
The objective of this work was to prepare benzofuroxan derivatives as new, dense, potentially ene... more The objective of this work was to prepare benzofuroxan derivatives as new, dense, potentially energetic materials and to investigate their properties, with the main focus being on 5,6-dinitrobenzofuroxan (5,6-DNBF). 5,6-DNBF was prepared by a 3-step reaction sequence: a) 1-azido-3-nitrobenzene was synthesized by diazotation of 3-nitroaniline with sodium nitrite and subsequent reaction with sodium azide in acetic/sulfuric mixed acids; b) it was nitrated with HNO3/H2SO4 to 1-azido-2,4,5-trinitrobenzene; c) thermal cyclization of the latter compound in a polar solvent gave the desired 5,6-DNBF (m.p. 177 °C). It was fully characterized by UV/VIS, FT-IR and NMR spectroscopy, mass spectrometry and single crystal X-ray diffraction. The density of the compound (X-ray) was found to be comparatively high (1.88 g/cm3), and to be superior to the previously known, isomeric energetic material − 4,6-dinitrobenzofuroxan (4,6-DNBF) (1.76 g/cm3). Furthermore, the synthesis of some other benzofuroxan derivatives, potentially interesting as high energy, density materials (HEDMs), has been carried out. The densities of the compounds obtained were calculated using ACD Labs software (version 4.0). Based on the results obtained, it could be concluded that 5,6-DNBF is one of the densest nitro derivatives of the benzofuroxan series, comparable to CL-14, CL-17, CL-18, and thus could have potential applications as an HEDM.
Журнал аналитической химии, 2016
A low cost and simply fabricated amperometric glucose biosensor based on glucose oxidase (GOx) an... more A low cost and simply fabricated amperometric glucose biosensor based on glucose oxidase (GOx) and 1,10 phenanthroline 5,6 dione (PD) modified graphite rod electrode was developed. The elec trode exhibited good electrocatalytic activity with a well defined hyperbolic dependence of amperometric signal upon glucose concentration in the presence and in the absence of oxygen. The electrode showed a good reproducibility and repeatability with relative standard deviation of less than 3 and 5%, respectively. The interaction of GOx with PD mediator was confirmed by means of UV⎯Vis absorbance spectroscopy.
Bioorganic & Medicinal Chemistry Letters, 2016
Naphtho[1',2&... more Naphtho[1',2':4,5]imidazo[1,2-a]pyridine-5,6-diones (NPDOs), a new type of N-heterocycle-fused o-quinones, have been synthesized. They have been found to be efficient electron-accepting substrates of NADPH-dependent single-electron-transferring P-450R and two-electron transferring NQO1, generating reactive oxygen species (ROS) with a concomitant decrease in NADPH, which is consistent with redox-cycling. The reactivity of NPDOs toward P-450R (in terms of kcat/Km) varied in the range of 10(6)-10(7)M(-1)s(-1), while their reduction by NQO1 proceeded much faster, approaching the diffusion control limit (kcat/Km∼10(8)-10(9)M(-1)s(-1)). NPDOs exhibited relatively high cytotoxic activity against human lung carcinoma (A-549) and breast tumor (MCF-7) cell lines (LC50=0.1-8.3μM), while promyelocytic leukemia cells (HL-60) were less sensitive to NPDOs (LC50⩾10μM). 3-Nitro-substituted NPDO (11) revealed the highest potency against both A-549 and MCF-7 cell lines, with LC50 of 0.12±0.03μM and 0.28±0.08μM, respectively. Dicoumarol partly suppressed the activity of the compounds against A-594 and MCF-7 cell lines, suggesting that their cytotoxic action might be partially influenced by NQO1-mediated bioreductive activation.
Ecotoxicology of Explosives, 2009
Methods in Enzymology, 2004
Publisher Summary This chapter analyzes the structure-activity relationships in two-electron redu... more Publisher Summary This chapter analyzes the structure-activity relationships in two-electron reduction of quinines. Quinones may accept electrons from various flavoenzymes, iron-sulfur proteins and photosynthetic reaction centers. The energetics of the quinine reduction are studied extensively by pulse-radiolysis, electron spin resonance, and electrochemical techniques. The single-electron reduction of quinones by flavoenzyme dehydrogenases-electrontransferases may be treated according to an ‘‘outer-sphere electron transfer’’ model. In general, the reaction rates increase with an increase in quinine single-electron reduction potential. In this chapter, it is demonstrated that multiparameter regression analysis, by using redox potential and several simple structural parameters of quinones, may provide important information on the mechanisms of two-electron enzymatic reduction. In view of the simplicity of the single-electron reduction mechanism and the presumable involvement of single-electron transfers in two-electron reduction, the single-electron reduction of quinones is analyzed. Single-electron reduction of quinones by flavoenzymes is analyzed. The chapter presents the outer-sphere electron transfer model in single-electron reduction of quinones. Structure-activity relationships in single-electron reduction of quinones by NADPH:Cytochrome P-450 reductase and ferredoxin:NADP + reductase are explored. Two-electron reduction of quinones by flavoenzymes is explained in the chapter. The chapter describes the mechanism of two-electron (Hydride) transfer.
FEBS Journal, 2008
The organic nitrate esters glycerol trinitrate (GTN), pentaerythritol tetranitrate (PETN) and the... more The organic nitrate esters glycerol trinitrate (GTN), pentaerythritol tetranitrate (PETN) and their analogues (Fig. ) are extensively used as explosives [1-3]. They may be considered as important environmental pollutants because of the production of large amounts of wastewater during their manufacture, and soil
Journal of Solid State Electrochemistry, 2014
In this study, two ortho-quinoidal compounds, 1,10-phenanthroline-5,6-dione (PD) and 9,10-phenant... more In this study, two ortho-quinoidal compounds, 1,10-phenanthroline-5,6-dione (PD) and 9,10-phenanthrenequinone (PQ), were examined as electron transfer mediators suitable for amperometric glucose biosensors. The dependences of the electrochemical responses of PD- and PQ-based amperometric glucose biosensors on varied concentrations of glucose were investigated under aerobic and anaerobic conditions. The PD-modified graphite rod (GR) electrode revealed a current response seven times higher than that of the PQ-modified GR electrode. The reactivity indices of ortho-quinoidals assessed by means of B3LYP functional method applying 6-311G(D) basis set showed that the electron-accepting potency for PD was markedly higher as compared with that of PQ. Compared to PQ, considerably higher reactivity of PD has been defined in the reactions with NADP+-ferredoxin reductase (FNR, EC 1.18.1.2) as a model single-electron transfer FAD-dependent enzyme, which provided an additional evidence for PD as a more efficient mediator compared to PQ. This study illustrates that PD can be applied as a redox mediator for glucose oxidase and it could be more suitable for a reagent-less biosensor design than PQ.
Journal of Biological Chemistry, 2003
Mammalian thioredoxin reductases (TrxR) are important selenium-dependent antioxidant enzymes. Qui... more Mammalian thioredoxin reductases (TrxR) are important selenium-dependent antioxidant enzymes. Quinones, a wide group of natural substances, human drugs, and environmental pollutants may act either as TrxR substrates or inhibitors. Here we systematically analyzed the interactions of TrxR with different classes of quinone compounds. We found that TrxR catalyzed mixed single-and two-electron reduction of quinones, involving both the selenium-containing motif and a second redox center, presumably FAD. Compared with other related pyridine nucleotide-disulfide oxidoreductases such as glutathione reductase or trypanothione reductase, the k cat /K m value for quinone reduction by TrxR was about 1 order of magnitude higher, and it was not directly related to the one-electron reduction potential of the quinones. A number of quinones were reduced about as efficiently as the natural substrate thioredoxin. We show that TrxR mainly cycles between the four-electron reduced (EH 4 ) and two-electron reduced (EH 2 ) states in quinone reduction. The redox potential of the EH 2 /EH 4 couple of TrxR calculated according to the Haldane relationship with NADPH/NADP ؉ was ؊0.294 V at pH 7.0. Antitumor aziridinylbenzoquinones and daunorubicin were poor substrates and almost inactive as reversible TrxR inhibitors. However, phenanthrene quinone was a potent inhibitor (approximate K i ؍ 6.3 ؎ 1 M). As with other flavoenzymes, quinones could confer superoxide-producing NADPH oxidase activity to mammalian TrxR. A unique feature of this enzyme was, however, the fact that upon selenocysteine-targeted covalent modification, which inactivates its normal activity, reduction of some quinones was not affected, whereas that of others was severely impaired. We conclude that interactions with TrxR may play a considerable role in the complex mechanisms underlying the diverse biological effects of quinones. Thioredoxin reductase (TrxR, 1 EC 1.8.1.9) catalyzes NADPHdependent reduction of the redox-active disulfide in thioredoxin (Trx), which serves a wide range of functions in cellular proliferation and redox control (1, 2). Thioredoxin reductases are homodimeric proteins that differ in properties between different classes of organisms. Low M r (34-kDa subunit) TrxRs of prokaryotes, plants, or yeast contain FAD and a redox-active disulfide/dithiol active site and display narrow substrate specificities. High M r (54 -58 kDa) TrxRs of animals have in contrast remarkably wide substrate specificities, explained by an additional easily accessible C-terminal redox center. This redox center is either a disulfide/dithiol as in TrxR of Plasmodium falciparum or Drosophila melanogaster or a selenocysteinecontaining selenenylsulfide/selenolthiol motif as found in TrxRs of mammals . In recent years, the catalytic mechanism of mammalian TrxR has been unraveled in significant detail. The three-dimensional crystal structure of rat TrxR is similar to that of glutathione reductase, including conserved FAD and NADP(H)-binding domains, but TrxR has a 16-residue C-terminal extension carrying the catalytic Cys-497/Sec-498 couple that in essence substitutes for glutathione as a substrate of the N-terminally located active site disulfide/dithiol motif (7). In the catalytic cycle of mammalian TrxR, NADPH first reduces FAD, which subsequently passes redox equivalents to the redox-active disulfide with formation of a dithiol, located within a conserved -CVNVGC-sequence. Finally, this dithiol reduces the selenenylsulfide formed by the Cys and Sec residues in a -GCUG sequence located at the C-terminal end of the other subunit in the dimeric enzyme . The so-formed selenolthiol is the proper active site of mammalian TrxR, reducing Trx or other substrates such as lipoic acid, ascorbic acid, or the synthetic model substrate, 5,5Ј-dithiobis-(2-nitrobenzoic acid) (DTNB) (9). Consecutive reduction of the three redox-active motifs of mammalian TrxR, i.e. the FAD, the N-terminal disulfide, and the C-terminal selenenylsulfide, gives two-, four-, and six-electron reduced states of the enzyme, with specific spectral properties that are well characterized (5, 8). It is believed that during normal catalysis, mammalian TrxR cycles between the two-and fourelectron reduced states with the two or four electrons shared mainly between the catalytic disulfide and the selenenylsulfide (5). The disulfide/dithiol motif also forms a charge transfer complex with the FAD (8). Recently, this mechanism was demonstrated also for D. melanogaster TrxR where, however, a
IUBMB Life, 1998
In order to determine the rote of glutathione reductase (GR) in protection against AI‐phtalocyani... more In order to determine the rote of glutathione reductase (GR) in protection against AI‐phtalocyanine tetrasulfonate‐sensitized human erythrocyte photolysis, we have studied the effects of antitumour alkylating agents that inactivate GR, on photohemolysis rate. The rates of inactivation of reduced GR decreased in order BCNU > pharanox (N‐p‐[bis‐(2‐chloroethyl)‐amino]‐phe‐nylacetic acid N‐oxide) > phenalol (N‐p‐[bis‐(2‐chloroethyl)‐amino]‐phenylacetyl‐L‐phenylalanine) > o‐F‐ and p‐F‐lophenal (o‐ and p‐isomers of N‐p‐[bis‐(2‐chloroethyl)‐amino]‐phenylacetyl‐D,L‐fluorophenylalanine) > D,L‐melphalan. As supposed, erythrocyte photolysis was accelerated by BCNU and pharanox, however, it was slowed down by phenylalanine mustards. The latter effect was explained by singlet oxygen quenching and/or photooxidation reactions of these compounds. This points out to a possibility of certain phenylalanine derivatives to neutralize the side‐effects of photodynamic therapy.
FEBS Letters, 1998
Quantitative structure activity relationships (QSARs) for the conversion of nitrobenzimidazolones... more Quantitative structure activity relationships (QSARs) for the conversion of nitrobenzimidazolones and nitrobenzimidazoles by rat liver DT-diaphorase (EC 1.6.99.2) are described. The parameter used for description of the QSARs is the energy of the lowest unoccupied molecular orbital (E(LUMO)) of the nitroaromatic compounds. Interestingly, correlations with E(LUMO) were observed for both the natural logarithm of k t , but also for the natural logarithm of k t /K m . The minimal kinetic model in line with these QSARs is a ping-pong mechanism that includes a substrate binding equilibrium in the second half reaction.
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Papers by Zilvinas Anusevičius