Derivatives of Aminoquinones with N-protected Amino Acids

Summary The synthesis of derivatives of aminoquinones with N-protected amino acids is reported here. 2-Amino-1,4-benzoquinone and 2-amino-1,4-naphthoquinone, prepared by the azide method in yields of 60 and 95% respectively, were coupled with N-Boc-protected amino acids including glycine, serine, proline and tyrosine, to give the correspondening derivatives.N, N′-Diisopropylcarbodiimide/1-hydroxybenzotriazole orN, N′-dicyclohexylcarbodiimide/HOBt used as coupling reagents provided the expected products in satisfactory yields and purities as supported by TLC, HPLC and spectral analysis.     

Formation of electronically excited states during the interaction of p-benzoquinone with hydrogen peroxide

The reaction between p-benzoquinone and H₂O₂ in slightly alkaline solutions yields three major quinoid products that accumulate in the reaction mixture: (a) 2,3-epoxy-p-benzoquinone, (b) 2-hydroxy-p-benzoquinone and (c) p-benzohydroquinone. The reaction is accompanied by photoemission, probably originating from excited triplet 2-hydroxy-p-benzoquinone. These products originate from hydrogen peroxide and hydroxide nucleophilic addition to the C₂?C₃ double bond, as well as secondary redox interactions. The hydroxy substituent and the epoxide ring exert a substantial influence on the electronic distribution in the p-benzoquinone molecule leading to a decrease in the half-wave potential, as compared to the parent p-benzoquinone. The generation of electronically excited states is the result of reactions secondary to the nucleophilic additions involving 2-hydroxy-p-benzosemiquinone, H₂O₂ and hydroxyl radical. The process involves the primary oxidation of 2-hydroxy-p-benzosemiquinone by hydrogen peroxide, followed by oxidation of the semiquinone by hydroxyl radical leading to the formation of the electronically excited quinone. The decay of the excited triplet to the ground state is accompanied by photoemission with maximal intensity at 485–530 nm. Thermodynamic calculations along with an observed increase of photoemission intensity in anaerobiosis point to the triplet (n, π*) multiplicity of the excited state. The efficiency of chemiluminescence could be calculated as 10^?8 photons/2-hydroxy-p-benzoquinone molecule formed. Photoemission arising from the p-benzoquinone/H₂O₂ reaction was inhibited efficiently by addition of GSH to the reaction mixture. This may be due to deactivation of the triplet quinone by a 2-glutathionyl-p-benzohydroquinone adduct, involving thioether α-hydrogen atom-transfer to the triplet ketone.     

Quinones as External Electron Acceptors in Steroid Dehydrogenation with Entrapped Cells in Organic Medium

A series of quinone-based compounds were tested for their ability to act as external electron acceptors in the ¹-dehydrogenation of-α-methyl-hydrocortisone-21-acetate, with polyurethane-entrapped Arthrobacter simplex cells in buffer-saturated n-decan-1-ol. This organic solvent was needed to solubilize the steroid substrate. In aqueous medium, the conversion with free cells virtually stopped after one hour, probably due to substrate limitation. All the tested quinones acted as external electron acceptors, increasing the bioconversion rate. The process kinetics were complex. However, when keeping the concentration of one of the substrates (steroid or quinone) constant and varying that of the other, Michaelis-Menten kinetics provided a reasonably good model for the initial reaction rates, and apparent kinetic constants were estimated. The most effective of the tested external electron acceptors were 2,6-dimethyl-p-benzoquinone and menadione. Mass transfer limitations seemed to appear after some hours of reaction, with low concentrations of the more efficient quinones, when the biocatalyst microenvironment was quinone- and possibly oxygen-depleted. Monosodium glutamate was included with the cells in the immobilisation foam, as an activity-stabilizing agent. It was observed that some of the quinones apparently formed complexes with this glutamate, thereby influencing the kinetics of the process. The catalytic half-life of the system depended on the quinone concentration and optimal values (60-80 h) were observed at 1 mM levels of 2,6-dimethyl-p-benzoquinone or menadione. Quinone toxicity, direct or through the formation of peroxides in the aerobic reoxidation process, may be at the origin of enzyme deactivation.     

Flash oxygen yield patterns of autotrophically and photoheterotrophically grown Chlamydobotrys stellata in the presence and absence of lipophilic acceptors

The obligate phototrophic green alga Chlamydobotrys stellata does not evolve oxygen when grown in CO₂-free atmosphere on acetate. With the application of the lipophilic acceptor 2,6-dichloro-p-benzoquinone it was investigated whether this phenomenon is caused by the inactivation of the water-splitting system or by an inhibition of the electron transport chain. It was found that in the presence of DCQ, the photoheterotrophic alga exhibited a normal period-4 flash oxygen pattern, but the steady state yield was only 25% of that measured in the autotrophic cells. After DCQ addition, the initial distribution of S-states and the values of the transition probabilities proved to be the same in the autotrophic and photoheterotrophic algae. These results indicate that photoheterotrophic growth conditions inhibit the electron transport of Chl. stellata behind the acceptor site of DCQ, but the water-splitting system remains active with a reduced oxygen evolving capacity.Abbreviations Chl chlorophyll - DCQ 2,6-dichloro-p-benzoquinone - DCMU 3-(3,4)-dichlorophenyl)-1,1-dimethylurea - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - pBQ 1,4-benzoquinone - PS I photosystem I - PS II photosystem II     

Electrocatalysis with a Glucose-Oxidase-immobilized Graphite Electrode

Glucose oxidase was immobilized on the surface of a graphite electrode by irreversible adsorption. An electrocatalytic steady-state current for the oxidation of D-glucose was observed using this electrode in the presence of p-benzoquinone as an electron transfer mediator. The electrocatalytic current at 0.5 V vs. SCE was analyzed as a function of the concentrations of D-glucose and p-benzoquinone, and the maximum current, I_s^max, and the Michaelis constants (K₁ and K₂ for D-glucose and p-benzoquinone, respectively) of the electrocatalysis were determined. The dependence of the current on the electrode potential, pH, and temperature was also investigated. The results indicate that the kinetics of the immobilized enzyme are essentially the same as those of the enzyme in the solubilized state. The effect of various electron transfer mediators on the electrocatalytic current was also examined and evaluated in terms of I_s^max, K₁, and K₂ values.     

Histone propinquity using imidoesters.

Native chromatin was reacted with bifunctional protein cross-linking reagents of varying molecular sizes. Short cross-linkers do not produce significant yields of polymeric histones whereas intermediate-sized materials such as dimethyladipimidate produce extensive interaction between histones. Dimethylsuberimidate reacts with all histone fractions but only generates polymers of F₁ and of F_2b and F_2a2.     

Purification and properties of a polyamine oxidase from Zea mays

Polyamine oxidase, purified 260-fold from maize shoots, was light yellow in colour. Maximum light-absorption was at 450 nm and was decreased by the addition of either sodium dithionite or spermidine, but not by putrescine. Under aerobic conditions, the enzyme could use p-benzoquinone as an electron acceptor. Cu²⁺ inhibited the enzyme activity, while SO₃⁻ was stimulatory. Several metal-binding agents and thiol reagents were without effect.     

Characterization of photosystem II in stroma thylakoid membranes

The functional state of the PS II population localized in the stroma exposed non-appressed thylakoid region was investigated by direct analysis of the PS II content of isolated stroma thylakoid vesicles. This PS II population, possessing an antenna size typical for PS II, was found to have a fully functional oxygen evolving capacity in the presence of an added quinone electron acceptor such as phenyl-p-benzoquinone. The sensitivity to DCMU for this PS II population was the same as for PS II in control thylakoids. However, under more physiological conditions, in the absence of an added quinone acceptor, no oxygen was evolved from stroma thylakoid vesicles and their PS II centers were found to be incapable to pass electrons to PS I and to yield NADPH. By comparison of the effect of a variety of added quinone acceptors with different midpoint potentials, it is concluded that the inability of PS II in the stroma thylakoid membranes to contribute to NADPH formation probably is due to that Q_A of this population is not able to reduce PQ, although it can reduce some artificial acceptors like phenyl-p-benzoquinone. These data give further support to the notion of a discrete PS II population in the non-appressed stroma thylakoid region, PS II, having a higher midpoint potential of Q_A than the PS II population in the appressed thylakoid region, PS II. The physiological significance of a PS II population that does not produce any NADPH is discussed.Abbreviations pBQ p-benzoquinone - Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCIP 2,6-dichloroindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMBQ 2,5-dimethyl-p-benzoquinone - DQ duroquinone(tetramethyl-p-benzoquinone) - FeCN ferricyanide (potassium hexacyanoferrat) - MV methylviologen - NADPH,NADP⁺ reduced or oxidized form of nicotinamide adenine dinucleotide phosphate respectively - PpBQ phenyl-p-benzoquinone - PQ plastoquinone - PS II photosystem II - PS I photosystem I - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - E microEinstein     

Glucosamine-mediated detoxification of p-benzoquinone and its removal by chitosan

p-Benzoquinone non-enzymatically reacted with d-glucosamine at physiological pH and moderate temperature. The reaction of p-benzoquinone with glucosamine was signaled by changes in the UV and visible spectra. The reactivity proceeded fastest at pH values above 7, with a sharp drop from pH 6.5 to 7.0, and the reaction was negligible in acidic conditions. The order of reactivity of amino sugars was d-mannosamine > d-glucosamine > d-galactosamine. From the reaction mixture, four conversion products were isolated and none was toxic to Escherichia coli even at 500–700 g ml^–1, while p-benzoquinone was cytotoxic to E. coli at 20 g ml^–1. Chitosan could react with p-benzoquinone efficiently and remove this toxicant in aqueous solution.     

Defensive secretion of the tenebrionid beetle,Blaps mucronata: Physical and chemical determinants of effectiveness

Summary The primary components of the defensive secretions ofBlaps mucronata (Tenebrionidae) are two quinones (methyl-p-benzoquinone and ethyl-p-benzoquinone) and the hydrocarbon 1p-n-tridecene. The hydrocarbon is shown, by comparison with longer- and shorter-chainn-alkanes and 1-n-alkenes, to be optimally suited as carrier of the quinones, and as a surfactant that promotes spread of secretion over the beetle's body following discharge from the gland openings at the abdominal tip. As shown from repellency tests with ants (Monomorium pharaonis) and topical irritancy tests with cockroaches (Periplaneta americana), the antiinsectan potency of the secretion derives as much from the hydrocarbon as from the quinones.Paper no. 82 of the seriesDefensive Mechanisms of Arthropods     

Dechlorination of tetrachloroguaiacol by laccase of white-rot basidiomycete Coriolus versicolor

 Degradation of tetrachloroguaiacol is catalyzed by an extracellular enzyme, the laccase of the white-rot fungus Coriolus versicolor. This enzyme catalyzes the dechlorination of tetrachloroguaiacol and release of chloride ions. The pathway for the degradation was deduced from the intermediates produced by purified laccase and ¹⁸O-labeling experiments. The first step is demethylation. The resulting tetrachlorocatechol is dechlorinated to give 2,3,5-trichloro-6-hydroxy-p-benzoquinone, 2,5-dichloro-3,6-dihydroxy-p-benzoquinone, and dichloro-6-hydroxy-p-benzoquinone. Isotopic experiments established the mechanism of dechlorination of tetrachloroguaiacol by laccase. The laccase-catalyzed dechlorination is not caused by oxidative coupling but by nucleophilic substitution in which Cl^- is released by water from cation radicals generated by laccase. Received: 25 August 1995/Received revision: 27 October 1995/Accepted: 20 November 1995     

Structure of a New Opine,Mikimopine, in Hairy Root Induced by Agrobacterium rhizogenes

A enzyme that catalyzed the specific formation of ascorbic acid-2-phosphate (AsA2P) from ascorbic acid (AsA) and adenosine-5′-triphosphate (ATP), was purified 3,200-fold to homogeneity from a cell extract of Pseudomonas azotocolligans. The purified enzyme appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and consisted of a single polypeptide with a molecular weight of about 30,000. Of phosphoryl donors tested, p-nitrophenylphosphate (p-NPP) and pyrophosphate (PPi) were as effective as ATP. Optimal pHs for the phosphorylating activity were around 4.0 and 5.5 when PPi and ATP were used as phosphoryl donors, respectively. The Km for AsA was 147 mm. The enzyme activity was inhibited by Cu²⁺, but not by sulfhydryl reagents.

The enzyme simultaneously had phosphatase activity at weakly acidic or neutral pH and the Km for p-NPP in the phosphatase activity was 0.38 mm. The enzyme was tentatively named “ascorbic acid phosphorylating enzyme.”     

Inhibition of jack bean urease by tetrachloro-o-benzoquinone and tetrachloro-p-benzoquinone

Tetrachloro-o-benzoquinone (TCoBQ) and tetrachloro-p-benzoquinone (TCpBQ) were studied as inhibitors of jack bean urease in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA, 25°C. The mechanisms of inhibition were evaluated by analysis of the progress curves obtained with two procedures: the reaction initiated by addition of the enzyme and the reaction initiated by addition of the substrate after preincubation of the enzyme with the inhibitor. The obtained results were characteristic of slow-binding inhibition. The effects of different inhibitor concentrations on the initial and steady-state velocities obeyed the relationships of two-step enzyme-inhibitor interaction, qualified as mechanism B. It was found that TCoBQ and TCpBQ are strong urease inhibitors. TCpBQ is more effective than TCoBQ with the overall inhibition constant of K_i* = 4.5 × 10^{? 7} mM. The respective inhibition constant of TCoBQ was equal to: K_i* = 2.4 × 10^{? 6} mM. The protective experiment proved that the urease active site is involved in the tetrachlorobenzoquinone inhibition process. High effectiveness of thiol protectors against inhibition by TCoBQ and TCpBQ indicates the strategic role of the active site sulfhydryl group in the blocking process. The stability of the complexes: urease-TCoBQ and urease-TCpBQ was tested in two ways: by dilution or addition of dithiothreitol. No recovery of urease activity bound in the urease-inhibitor complexes proves that the complexes are stable and strong.     

Immunocytochemical staining of cytocentrifuge prepared cultured cells: nonspecific staining and its elimination

Summary In an attempt to localize hormones in cytocentrifuge-prepared cultured cells of small cell carcinoma of the lung (SCCL), various modifications of the immunoperoxidase (PAP) procedure (Sternberger, 1979) were tested. When using glutaraldehyde, formaldehyde, orp-benzoquinone fixation (Pearse & Polak, 1975) and rabbit antibodies in primary or bridging steps of the PAP procedure, nonspecific staining (false positives) could be elicited with the majority of rabbit antibodies tested, but not with antibodies from other animal sources. This problem could be eliminated by fixation of cells either with formalin-acetone (Masonet al., 1975) or, when using antibodies from a source other than rabbit, glutaraldehyde. It was not possible to localize ACTH in DMS-79, a human SCCL line known to produce this hormone. However, calcitonin was localized in the calcitonin-producing SCCL line DMS-53. Failure to localize ACTH in DMS-79 may be due to the lower levels of this hormone in DMS-79, as compared to the levels of calcitonin in DMS-53. This study emphasizes the importance of proper controls before concluding successful localization in a given immunocytochemical preparation of cultured cells.     

Genome‐wide identification of tolerance mechanisms toward p‐coumaric acid in Pseudomonas putida

The soil bacterium Pseudomonas putida KT2440 has gained increasing biotechnological interest due to its ability to tolerate different types of stress. Here, the tolerance of P. putida KT2440 toward eleven toxic chemical compounds was investigated. P. putida was found to be significantly more tolerant toward three of the eleven compounds when compared to Escherichia coli. Increased tolerance was for example found toward p‐coumaric acid, an interesting precursor for polymerization with a significant industrial relevance. The tolerance mechanism was therefore investigated using the genome‐wide approach, Tn‐seq. Libraries containing a large number of miniTn5‐Km transposon insertion mutants were grown in the presence and absence of p‐coumaric acid, and the enrichment or depletion of mutants was quantified by high‐throughput sequencing. Several genes, including the ABC transporter Ttg2ABC and the cytochrome c maturation system (ccm), were identified to play an important role in the tolerance toward p‐coumaric acid of this bacterium. Most of the identified genes were involved in membrane stability, suggesting that tolerance toward p‐coumaric acid is related to transport and membrane integrity.

     

The chemical mechanism of action of glucose oxidase from Aspergillus niger

Glucose oxidase from Aspergillus niger (EC 1.1.3.4) is able to catalyze the oxidation of -D-glucose with p-benzoquinone, methyl-1,4-benzoquinone, 1,2-naphthoquinone, 1,2-naphthoquinone-4-sulfonic acid, potassium ferricyanide, phenazine methosulfate, and 2,6-dichloroindophenol. In this work, the steady-state kinetic parameters, V ₁/K _B , for reactions of these substrates were collected from pH 2.5–8. Further, the molecular models of the enzyme's active site were constructed for the free enzyme in the oxidized state, the complex of -D-glucose with the oxidized enzyme, the complex of reduced enzyme with methyl-1,4-benzoquinone, the reduced enzyme plus 1,2-naphthoquinone-4-sulfonic acid, oxidized enzyme plus reduced 1,2-naphthoquinone-4-sulfonic acid (hydroquinone anion), and oxidized enzyme plus fully reduced 1,2-naphthoquinone-4-sulfonic acid.Combining the steady-state kinetic and structural data, it was concluded that Glu412 bound to His559, in the active site of enzyme, modulates powerfully its catalytic activity by affecting all the rate constants in the reductive and the oxidative half-reaction of the catalytic cycle. His516 is the catalytic base in the oxidative and the reductive part of the catalytic cycle. It was estimated that the pK _a of Glu412 (bound to His559) in the free reduced enzyme is 3.4, and the pK _a of His516 in the free reduced enzyme is 6.9.     

The formation and characterization of copper(II) 1,4-benzenediolate and p-semiquinonate complexes

Strongly oxidizing p-quinones such as tetrachloro-1,4-benzoquinone and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone undergo stepwise oxidative addition reactions with copper(I) chloride and bromide in pyridine resulting in copper(II) p-semiquinone and dinuclear copper(II) 1,4-benzenediolate pyridine complexes.     

The cyclic electron pathways around photosystem I in Chlamydomonas reinhardtii as determined in vivo by photoacoustic measurements of energy storage

The photoacoustic technique was used to measure energy storage by cyclic electron transfer around photosystem I in intact Chlamydomonas reinhardtii cells illuminated with far-red light (>715 nm). The in-vivo cyclic pathway was characterized by investigating the effects of various chemicals on energy storage. Participation of plastoquinone and ferredoxin in the cyclic electron flow was confirmed by the complete suppression of energy storage in the presence of the plastoquinol antagonist 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and the ferredoxin inhibitors/competitors methylviologen, phenylmercuric acetate and p-benzoquinone. Two alternative electron cycles are demonstrated to operate in vivo. One cycle is sensitive to antimycin A, myxothiazol and 2-(n-heptyl)-4-hydroxyquinoline N-oxide (HQNO) and is catalyzed by ferredoxin which reduces plastoquinone through a route involving cytochrome b ₆ and its protonmotive Q-cycle. The other cycle is unaffected by the above-mentioned inhibitors but is sensitive to N-ethylmaleimide (NEM), an inhibitor of the ferredoxin-NADP reductase, and 2-monophosphoadenosine-5-diphosphoribose (PADR), an analogue of NADP, showing that the electron recycling was mediated by NADPH. Possibly, electrons enter the plastoquinone pool through the action of a NAD(P)H dehydrogenase, which is insensitive to classical inhibitors of the mitochondrial NADH dehydrogenase. Loss of energy storage by photosystem-I-driven cyclic electron transfer in farred light was observed only when antimycin A, myxothiazol or HQNO was used in combination with NEM or PADR. Analysis of the light-intensity dependence and the rate of in-vivo cyclic electron transfer in the presence of various inhibitors indicates that the NADPH-dependent electron-cycle is the preferential cyclic pathway in Chlamydomonas cells illuminated with far-red light.Abbreviations A^max maximal photothermal signal - Cyt cytochrome - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - ES photochemical energy storage - FNR ferredoxin NADP⁺ reductase - HQNO 2-(n-heptyl)-4-hydroxyquinoline N-oxide - NEM N-ethylmaleimide - P₇₀₀ reaction-center pigment of PSI - PADR 2-monophosphoadenosine-5-diphosphoribose - pBQ p-benzoquinone - PMA phenylmercuric acetate We are very grateful to Dr. M.-H. Montane (Cadarache, Saint-Paul-lez-Durance, France) for her advice in the electroporation experiments.     

Circadian rhythmicity in the stimulation of bioluminescence by biogenic amines and MAO inhibitors inGonyaulax polyedra

In the dinoflagellateGonyaulax polyedra bioluminescence was investigated in constant darkness. Light emission was stimulated considerably and specifically by the biogenic smines epinephrine, 5-methoxytryptamine, and kynuramine. Various analogues and motabolites of these substances, such as norepinephrine, isoproterenol, phenylephrine, synephrine, metanephrine isoproterenol, phenylephrine, synephrine, metanephrine dopamine, 3,4-dihydroxymandelic and 3-methoxy hydroxymandelic acids, serotonin, N-acetylserotonin, melatonin, 5-hydroxytryptophol, 5-methoxytryptophol, kynurenine, 4-hydroxyquinoline, 3-hydroxyanthrani ic, and quinolinic acids were much less effective. Strong enhancement of bioluminescence, in the range of those obtained with the three stimulatory biogenic amines was also observed after administration of several compeunds acting as MAO inhibitors in mammalian systems, in particular, pargyline, amitriptyline,p-benzoquinone, tranylcypromine, harmaline, and noreleagnine. The responsiveness of cells towards epinephrine, 5-methoxytryptamine, kynuramine, amitriptyline,p-benzoquinone, and noreleagnine varied considerably within the circadian cycle, with the highest stimulations obtained during subjective night. These rhythms can be only partially explained by periodic bioluminescence capacity, and seem to comprise a cyclicity in the sensitivity of cells to the compounds mentioned.Paper presented at the 12th International Congress of Biometeorology (Vienna 1990)