Vectorial redox reactions of physiological quinones. I. Requirement of a minimum length of the isoprenoid side chain.

Physiological quinones carrying isoprenoid side chains have been compared with homologues lacking the side chain, for their ability to carry electrons and protons from dithionite to ferricyanide, trapped in liposomes. Six differential observations were made: (1) Plastoquinone and ubiquinones, with a side chain of more than two isoprene units, are by far better mediators than their short-chain homologues. Also other benzoquinones lacking a long side chain are poor catalysts, except dimethyl-methylenedioxy-p-benzoquinone, a highly autooxidizable compound. Tocopherol is a good catalyst. (2) Vitamin K-1 and K-2 are poor mediators compared to vitamin K-3. (3) The reaction catalyzed by quinones carrying long isoprenoid side chains has an about three-fold higher activation energy, irrespective of the catalytic efficiency. (4) The reaction catalyzed by quinones lacking a long side chain follows pseudo first-order kinetics, while the reaction with quinones carrying a long side chain is of apparently higher order. (5) The rate with ubiquinone-1 is increasing pH, while with ubiquinone-9 it is decreasing. (6) The reaction mediated by short-chain quinones seems to be satuarated at lower dithionite concentration. We conclude that isoprenoid quinones are able to translocate electrons and protons in lipid membranes, and that the side chain has a strong impact on the mechanism. This and the relevance of the model reaction for electron and proton transport in photosynthesis and respiration is discussed.     

Marine polyprenylated hydroquinones, quinones, and chromenols with inhibitory effects on leukotriene formation

A series of polyprenylated hydroquinones, quinones, and chromenols were isolated from the extracts of the marine sponge Ircinia spinosula and the brown alga Taonia atomaria, which gave rise to the constituents 1-4 and 5-8, respectively. Compounds 1, 2, 6, and 7 are new natural products, which were fully characterized. Their anti-inflammatory activities in terms of leukotriene formation were evaluated in an in vitro assay with pork leukocytes. The new hydroxylated compound, 2'-[28-hydroxy]heptaprenyl-1',4'-hydroquinone (= 2-[(2E,6E,10E,14E,18Z,22E)-19-(hydroxymethyl)-3,7,11,15,23,27-hexamethyloctacosa-2,6,10,14,18,22,26-heptaen-1-yl]benzene-1,4-diol; 1), the known tetraprenyl benzoquinone sargaquinone (5), and the known polyprenyl chromenols 3 and 4 exhibited the highest anti-inflammatory activities, with IC50 values of 1.9-9.4 microM (Table 3). Potential structure-activity relationships (SAR) are discussed.     

Occurrence, biosynthesis and function of isoprenoid quinones

Isoprenoid quinones are one of the most important groups of compounds occurring in membranes of living organisms. These compounds are composed of a hydrophilic head group and an apolar isoprenoid side chain, giving the molecules a lipid-soluble character. Isoprenoid quinones function mainly as electron and proton carriers in photosynthetic and respiratory electron transport chains and these compounds show also additional functions, such as antioxidant function. Most of naturally occurring isoprenoid quinones belong to naphthoquinones or evolutionary younger benzoquinones. Among benzoquinones, the most widespread and important are ubiquinones and plastoquinones. Menaquinones, belonging to naphthoquinones, function in respiratory and photosynthetic electron transport chains of bacteria. Phylloquinone K₁, a phytyl naphthoquinone, functions in the photosynthetic electron transport in photosystem I. Ubiquinones participate in respiratory chains of eukaryotic mitochondria and some bacteria. Plastoquinones are components of photosynthetic electron transport chains of cyanobacteria and plant chloroplasts. Biosynthetic pathway of isoprenoid quinones has been described, as well as their additional, recently recognized, diverse functions in bacterial, plant and animal metabolism.     

Synthesis of novel heterobranched beta-cyclodextrins having beta-D-N-acetylglucosaminyl-maltotriose on the side chain

From a mixture of N-acetylglucosaminyl-beta-cyclodextrin (GlcNAc-betaCD) and lactose, beta-D-galactosyl-GlcNAc-betaCD (Gal-GlcNAc-betaCD) was synthesized by the transfer action of beta-galactosidase. GlcNAc-maltotriose (Glc3) and Gal-GlcNAc-Glc3 were produced with hydrolysis of GlcNAc-betaCD by cyclodextrin glycosyltransferase, and Gal-GlcNAc-betaCD by bacterial saccharifying alpha-amylase respectively. Finally, GlcNAc-Glc3-betaCD and Gal-GlcNAc-Glc3-betaCD were synthesized in 5.2% and 3.5% yield when Klebsiella pneumoniae pullulanase was incubated with the mixture of GlcNAc-Glc(3) and betaCD, or Gal-GlcNAc-Glc3 and betaCD respectively. The structures of GlcNAc-Glc3-betaCD and Gal-GlcNAc-Glc3-betaCD were analyzed by FAB-MS and NMR spectroscopy and identified as 6-O-alpha-(6(3)-O-beta-D-N-acetylglucosaminyl-maltotriosyl)-betaCD, and 6-O-alpha-(4-O-beta-D-galactopyranosyl-6(3)-O-beta-D-N-acetylglucosaminyl-maltotriosyl)-betaCD respectively.     

Metabolic engineering of coenzyme Q by modification of isoprenoid side chain in plant

Coenzyme Q (CoQ), an electron transfer molecule in the respiratory chain and a lipid-soluble antioxidant, is present in almost all organisms. Most cereal crops produce CoQ9, which has nine isoprene units. CoQ10, with 10 isoprene units, is a very popular food supplement. Here, we report the genetic engineering of rice to produce CoQ10 using the gene for decaprenyl diphosphate synthase (DdsA). The production of CoQ9 was almost completely replaced with that of CoQ10, despite the presence of endogenous CoQ9 synthesis. DdsA designed to express at the mitochondria increased accumulation of total CoQ amount in seeds.     

Ubiquinone. Biosynthesis of quinone ring and its isoprenoid side chain. Intracellular localization

Ubiquinone, known as coenzyme Q, was shown to be the part of the metabolic pathways by Crane et al. in 1957. Its function as a component of the mitochondrial respiratory chain is well established. However, ubiquinone has recently attracted increasing attention with regard to its function, in the reduced form, as an antioxidant. In ubiquinone synthesis the para-hydroxybenzoate ring (which is the derivative of tyrosine or phenylalanine) is condensed with a hydrophobic polyisoprenoid side chain, whose length varies from 6 to 10 isoprene units depending on the organism. para-Hydroxybenzoate (PHB) polyprenyltransferase that catalyzes the condensation of PHB with polyprenyl diphosphate has a broad substrate specificity. Most of the genes encoding (all-E)-prenyltransferases which synthesize polyisoprenoid chains, have been cloned. Their structure is either homo- or heterodimeric. Genes that encode prenyltransferases catalysing the transfer of the isoprenoid chain to para-hydroxybenzoate were also cloned in bacteria and yeast. To form ubiquinone, prenylated PHB undergoes several modifications such as hydroxylations, O-methylations, methylations and decarboxylation. In eukaryotes ubiquinones were found in the inner mitochondrial membrane and in other membranes such as the endoplasmic reticulum, Golgi vesicles, lysosomes and peroxisomes. Still, the subcellular site of their biosynthesis remains unclear. Considering the diversity of functions of ubiquinones, and their multistep biosynthesis, identification of factors regulating their cellular level remains an elusive task.     

Studies of the location and function of isoprenoid quinones in chlorosomes from green sulfur bacteria

The chlorosome antenna of the green sulfur bacterium Chlorobium tepidum essentially consists of aggregated bacteriochlorophyll (BChl) c enveloped in a glycolipid monolayer. Small amounts of protein and the isoprenoid quinones chlorobiumquinone (CK) and menaquinone-7 (MK-7) are also present. Treatment of isolated chlorosomes from Cb. tepidum with sodium dodecyl sulfate (SDS) did not affect the quinones, demonstrating that these are located in a site which is inaccessible to SDS, probably in the interior of the chlorosomes. About half of the quinones were removed by Triton X-100. The non-ionic character of Triton probably allowed it to extract components from within the chlorosomes. MK-10 in chlorosomes from the green filamentous bacterium Chloroflexus aurantiacus was likewise found to be located in the chlorosome interior. The excitation transfer in isolated chlorosomes from Cb. tepidum is redox-regulated. We found a ratio of BChl c fluorescenceintensity under reducing conditions (Fred) to that under oxidizing conditions (Fox) of approximately 40. The chlorosomal BChl a fluorescence was also redox-regulated. When the chlorosomal BChl c–BChl c interactions were disrupted by 1-hexanol, the BChl c Fred/Fox ratiodecreased to approximately 3. When CK and MK-7 were extracted from isolated chlorosomes with hexane, the BChl c Fred/Fox ratio also decreased to approximately 3. A BChl c Fred/Fox ratio of 3–5 was furthermore observed in aggregates of pure BChl c and in chlorosomes from Cfx. aurantiacus which do not contain CK. We therefore suggest that BChl c aggregates inherently exhibit a small redox-dependent fluorescence (Fred/Fox 3) and that the large redox-dependent fluorescence observed in chlorobial chlorosomes (Fred/Fox 40) is CK-dependent.     

Synthesis and application of photoaffinity probe containing an intact isoprenoid chain

Two novel chemical probes each carrying an intact isoprenoid chain, a biotin tag and a benzophenone moiety were synthesized. Photoaffinity labeling of the Saccharomyces cerevisiae cell lysate revealed that these probes could selectively trap some proteins, and proteins with molecular weight of ～70 KDa appeared as a major band upon Streptavidin blot analysis.     

An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids

A simple small-scale procedure for the sequential extraction of isoprenoid quinones and polar lipids from bacterial cells was developed. Extraction with a biphasic mixture of petroleum ether (b.p. 60–80°C) and methanolic saline gave an upper phase containing isoprenoid quinones. The lower phase, containing the partially extracted organisms, was processed according to the Bligh and Dyer extraction method to give a polar lipid extract. As examples of the procedure, the isoprenoid quinones and polar lipids of Bacillus subtilis, Mycobacterium avium, Pseudomonas diminuta and Streptomyces griseus were extracted and analyzed.     

Synthesis of nucleotide oligomer having amino group in the side chain of internucleotidic bond

A convenient synthesis of d(TpT) derivative having amino group in the side chain of internucleotidic bond was achieved in a high yield using dichlorophosphoramidite as a phosphorylating reagent.     

Dendrocyin: an isocucurbitacin with novel cyclic side chain from Dendrosicyos socotrana

Dendrosicyos socotrana Balf.f. is a unique species (Cucurbitaceae) native to Socotra island in the horn of Africa. From the chloroform extract of the stems, A new isocucurbitacin (Dendrocyin) with unusual cyclization in the side chain; 24beta-ethoxy-20-25-epoxy-3alpha,16alpha-dihydroxy-9-methyl-19-norlanost-5(6) ene-2,11,22-trione has been isolated alongside isocucurbitacin R. Their structural configuration were established by usual spectroscopic (1H NMR, 13C NMR and DEPT) and two-dimensional NMR techniques (1H-1H Cosy, HMBC and HMQC).     

The distribution of isoprenoid quinones in streptococci of serological groups D and N.

The isoprenoid quinone contents of streptococci of serological groups D and N were investigated. Streptococcus faecalis, S. faecalis subsp. liquefaciens and S. faecalis subsp. zymogenes strains contained demethylmenaquinones with nine isoprene units as their major isoprenologues. Menaquinones with eight isoprene units predominated in S. faecium subsp. casseliflavus and S. faecium subsp. mobilis whereas menaquinones with nine isoprene units constituted the major components in strains of S. cremoris, S. cremoris subsp. alactosus, S. lactis and S. lactis subsp. diacetylactis. Strains of S. avium, S. bovis, S. durans, S. equinus, S. faecium, S. raffinolactis and S. suis contained neither menaquinones nor ubiquinones. The isoprenoid quinone data correlate well with other kinds of data on these organisms and are of value in the classification of these bacteria.     

Synthesis of new phorbol derivatives having ethereal side chain and evaluation of their anti-HIV activity

Several new phorbol derivatives having ethereal substituents at the 12-position were synthesized and subjected to biological evaluation to find new candidates of an anti-HIV agent. Among them, 12-O-(methoxymethyl)phorbol 13-decanoate showed potent inhibitory activity against infection of HIV-1 in MT-4 cells (EC50: 1.3 ng/mL) and relatively low cytotoxicity (CC50: 8.3 microg/mL). This compound was also found to have sufficient stability in mouse plasma compared with the corresponding 12-acetate derivative, which was an equipotent HIV-1 inhibitor, but with an activity that decreased considerably after plasma treatment.     

The nature, intergeneric distribution and biosynthesis of isoprenoid quinones and phenols in Gram-negative bacteria

1. Twenty-two aerobically grown Gram-negative bacteria were analysed for demethylmenaquinones, menaquinones, 2-polyprenylphenols, 6-methoxy-2-polyprenylphenols and ubiquinones. 2. All the eight enterobacteria and both the two facultative organisms (Aeromonas punctata and Aeromonas hydrophila) examined contain all the compounds listed above. The principal homologues are octaprenyl; in addition lower (down to tri- or tetra-prenyl for the 2-polyprenylphenols) and sometimes higher homologues are also present. 3. Strict aerobes are of two types, those that contain 2-polyprenylphenols, 6-methoxy-2-polyprenylphenols and ubiquinones, and those that contain ubiquinones only. The principal homologues are generally octa- or nona-prenyl, although one organism (Agrobacterium tumefaciens) has ubiquinone-10 as its principal homologue. As in the enterobacteria, lower homologues of these compounds are also present. 4. In Escherichia coli W, Pseudomonas ovalis Chester and Pseudomonas fluorescens, radioactivity from p-hydroxy[U-(14)C]benzoic acid is incorporated into 2-polyprenylphenols, 6-methoxy-2-polyprenylphenols, 6-methoxy-3-methyl-2-polyprenyl-1,4-benzoquinones, ubiquinones and a compound tentatively identified as 2-polyprenyl-1,4-benzoquinone. The fact that radioactivity is incorporated into the first three compounds suggests that in these organisms, and indeed in all those Gram-negative bacteria that contain 2-polyprenylphenols and 6-methoxy-2-polyprenylphenols, ubiquinones are formed by a biosynthetic sequence similar to that in Rhodospirillum rubrum. 5. The finding in ;Vibrio O1' (Moraxella sp.) and organism PC4 that 2-polyprenylphenols and 6-methoxy-2-polyprenylphenols are chemically and radiochemically undetectable leads to the conclusion that they are not intermediates in the biosynthesis of ubiquinone by these and by other Gram-negative bacteria that do not contain detectable amounts of 2-polyprenylphenols and 6-methoxy-2-polyprenylphenols. However, ;Vibrio O1' (organism PC4 was not examined) does contain 6-methoxy-3-methyl-2-polyprenyl-1,4-benzoquinone. 6. In Ps. ovalis Chester, radioactivity from l-[Me-(14)C]methionine is incorporated into the nuclear C-methyl and O-methyl groups of 6-methoxy-3-methyl-2-polyprenyl-1,4-benzoquinones and ubiquinone-9, and into the O-methyl group of 6-methoxy-2-polyprenylphenols.     

Main chain and side chain dynamics of oxidized flavodoxin from Cyanobacterium anabaena.

Oxidized flavodoxin from Cyanobacterium anabaena PCC 7119 is used as a model system to investigate the fast internal dynamics of a flavin-bearing protein. Virtually complete backbone and side chain resonance NMR assignments of an oxidized flavodoxin point mutant (C55A) have been determined. Backbone and side chain dynamics in flavodoxin (C55A) were investigated using (15)N amide and deuterium methyl NMR relaxation methods. The squared generalized order parameters (S(NH)(2)) for backbone amide N-H bonds are found to be uniformly high ( approximately 0.923 over 109 residues in regular secondary structure), indicating considerable restriction of motion in the backbone of the protein. In contrast, methyl-bearing side chains are considerably heterogeneous in their amplitude of motion, as indicated by obtained symmetry axis squared generalized order parameters (S(axis)(2)). However, in comparison to nonprosthetic group-bearing proteins studied with these NMR relaxation methods, the side chains of oxidized flavodoxin are unusually rigid.     

Formation of glutathione-conjugated semiquinones by the reaction of quinones with glutathione: an ESR study

The nonenzymatic reaction of the cytotoxic compounds menadione (2-methyl-1,4-naphthoquinone) and 1,4-naphthoquinone (a reactive metabolite of 1-naphthol) with reducing agents such as NADPH and glutathione led to the formation of semiquinone-free radicals, which were detected with electron spin resonance spectroscopy. In the presence of glutathione as a reducing agent, menadione and 1,4-naphthoquinone underwent net one-electron reduction and conjugation with glutathione. At higher concentrations of glutathione, 1,4-naphthoquinone formed the semiquinones of both the monoconjugate and the diconjugate. The naphthoquinone-glutathione conjugates should redox cycle in a manner already known for the menadione conjugate. The semiquinone intermediates could be detected only under a nitrogen atmosphere and are probably the primary oxygen-reactive species responsible for the redox cycling of menadione- and naphthoquinone-glutathione conjugates.