Heteroatom-Containing Antibacterial Phenolic Metabolites from a Terrestrial Ampelomyces Fungus

Two new sulfur-containing phenolic compounds, 7-hydroxy-5-hydroxymethyl-2H-benzo[1,4]thiazin-3-one (1) and 2,5-dihydroxy-3-methanesulfinylbenzyl alcohol (2), along with two known compounds, 3-chloro-2,5-dihydroxybenzyl alcohol (3) and 2-hydroxy-6-methylbenzoic acid (4), were isolated from the mycelial solid culture of a soil-derived Ampelomyces fungus by antibacterial assay-guided fractionation. Their structures were elucidated on the basis of spectroscopic analysis. Compounds 1–3 showed structure and microbial dependent antibacterial activities.     

降龙草的蒽醌类化学成分

为了解降龙草(Hemiboea subcapitata Clarke)的化学成分,从降龙草全草的乙醇提取物的石油醚和乙酸乙酯萃取部位分离得到了5个蒽醌类化合物。经波谱学分析鉴定为digiferruginol(1)、1,4-二羟基-2-羟甲基-9,10-蒽醌(2)、1,7-二羟基-2-羟甲基-9,10-蒽醌(3)、1-羟基-7-甲氧基-2-羟甲基-9,10-蒽醌(4)和1,4,7-三羟基-2-甲基-9,10-蒽醌(5)。其中化合物4和5为新化合物,其它3个化合物为首次从降龙草中分离得到。     

Evaluation of andrographolide-based analogs derived from Andrographis paniculata against Mythimna separata Walker and Tetranychus cinnabarinus Boisduval

To discover new natural-product-based pesticides, we structurally modified andrographolide, a labdane diterpenoid isolated from Andrographis paniculata, and stereoselectively prepared a series of 12α-(substituted)benzylamino-14-deoxyandrographolide derivatives (I–V). Three-dimensional structures of compounds 3c, 3d, IIIa and IIIb were further determined by single-crystal X-ray diffraction. Compounds IIa (R¹ = n-C₃H₇, R² = PhCH₂) exhibited more promising insecticidal activity against Mythimna separata than toosendanin. Compounds 3a (R¹ = H), Ib (R¹ = H, R² = 4-ClPhCH₂), and IVa (R¹ = 4-ClPh, R² = PhCH₂) showed potent acaricidal activity against Tetranychus cinnabarinus.     

Cyclopropane-based conformational restriction of GABA by a stereochemical diversity-oriented strategy: Identification of an efficient lead for potent inhibitors of GABA transports

A series of cyclopropane-based conformationally restricted γ-aminobutyric acid (GABA) analogs with stereochemical diversity, that is, the trans- and cis-2,3-methano analogs Ia and Ib and their enantiomers ent-Ia and ent-Ib, and also the trans- and cis-3,4-methano analogs IIa and IIb and their enantiomers ent-IIa and ent-Iib, were synthesized from the chiral cyclopropane units Type-a and Type-b that we developed. These analogs were systematically evaluated with four GABA transporter (GAT) subtypes. The trans-3,4-methano analog IIa had inhibitory effects on GAT3 (IC₅₀ = 23.9 μM) and betaine-GABA transporter1 (5.48 μM), indicating its potential as an effective lead compound for the development of potent GAT inhibitors due to its hydrophilic and low molecular weight properties and excellent ligand efficiency.     

Limitations of the lignin peroxidase system of the white-rot fungus, Phanerochaete chrysosporium

The lignin peroxidase enzyme system of the white-rot fungus, Phanerochaete chrysosporium was assayed for its capacity to degrade two recalcitrant aliphatic ether compounds, high-molecular-mass polyethylene glycol (PEG 20 000) and methyl tert-butyl ether. Ligninolytic cultures of Phanerochaete chrysosporium were spiked with each ether compound and incubated in reaction vessels. Separate incubations were conducted in which the ether compounds were present as sole carbon source. Other parameters, such as varying the methyl tert-butyl ether concentration and veratryl alcohol additions were tested. No significant degradation of either compound was observed under any of the conditions tested. Implications of these results are discussed with respect to the oxidative limitations of the lignin peroxidase enzyme system and structural features of substrate molecules that may be requisite for oxidation by this system.     

Scrutiny of chain-length and N-terminal effects in α-helix folding: a molecular dynamics study on polyalanine peptides

Protein folding remains an unsolved problem as main-chain, side-chain, and solvent interactions remain entangled and have been hard to resolve. Polyalanines are promising models to analyze protein folding initiation and propagation structurally as well as energetically. In the present work, the effect of chain-length and N-terminal residue stereochemistry in polyalanine peptides are investigated for their role in the nucleation of α-helical conformation. The end-protected polyalanine peptides, tetra-alanine, Ac-^LAla₄-NHMe (Ia) and Ac-^DAla-^LAla₃-NHMe (Ib), hexa-alanine, Ac-^LAla₆-NHMe (IIa) and Ac-^DAla-^LAla₅-NHMe (IIb), and octa-alanine, Ac-^LAla₈-NHMe (IIIa) and Ac-^DAla-^LAla₇-NHMe (IIIb), are assessed as chain-length and stereochemical-structure perturbed models. The appreciable variations in the sampling of α-helical conformation, including a sampling of α-helix folds, due to the cooperative effect of chain-length and N-terminal residue stereochemistry have been noted. The electrostatics of α-helical conformation rather than the conformational entropy of the main-chain appear to be decisive in the initiation of α-helix folding. The results of the present work will enhance our understanding on the nucleation of α-helical conformation in short peptides and aid in the design of novel peptides with α-helical structure that can modulate disease-related protein–protein interactions.     

华石斛化学成分研究

为了解华石斛(Dendrobium sinense)的化学成分,采用多种柱色谱技术从其全草乙醇提取液中分离纯化了10个化合物,经波谱分析分别鉴定为:鼓槌石斛素(1)、2′,4′-二羟基查尔酮(2)、2,5,7-三羟基-4-甲氧基-9,10-二氢菲(3)、4,7-二羟基-2,3-二甲氧基-9,10-二氢菲(4)、2,5-二羟基-3,4-二甲氧基-9,10-二氢菲(5)、2,7-二羟基-3,4,6-三甲氧基-9,10-二氢菲(6)、(E)松柏醛(7)、反式对羟基肉桂酸酯(8)、对羟基苯丙酸甲酯(9)和十二元内环酯(10)。所有化合物均为首次从华石斛中分离得到,其中化合物2、6、7和10对乙酰胆碱酯酶具有一定的抑制活性。     

Biosynthesis of the secondary metabolite veratryl alcohol in relation to lignin degradation in Phanerochaete chrysosporium

The lignin-degrading basidiomycete Phanerochaete chrysosporium synthesizes veratryl alcohol (3,4-dimethoxybenzyl alcohol) via phenylalanine, 3,4-dimethoxycinnamyl alcohol and veratrylglycerol. Study of the conversion of 3,4-dimethoxycinnamyl alcohol to veratrylglycerol and veratryl alcohol showed is to be (a) catalyzed by a secondary metabolic system, (b) markedly suppressed by culture agitation, and (c) strongly inhibited by l-glutamate. The amount of veratryl alcohol synthesized de novo was positively correlated with the O₂ concentration after primary growth. Other work has shown that the cinnamyl alcohol terminal residue in a lignin substructure model compound is degraded via arylglycerol and benzyl alcohol structures in ligninolytic cultures of P. chrysosporium, and that the ligninolytic system exhibits traits (a)-(c) above. Ligninolytic activity is also strongly and positively correlated with O₂ concentration. The results here suggest, therefore, that the actual biosynthetic secondary metabolic product is 3,4-dimethoxycinnamyl alcohol, but that this is degraded by the ligninolytic system to veratryl alcohol via veratrylglycerol. Veratryl alcohol is only slowly metabolized by the fungus, and accumulates.Non-standard abbreviation tlc thin layer chromatography     

Kinetics of Veratryl Alcohol Oxidation by Lignin Peroxidase and in situ Generated Hydrogen Peroxide in an Electrochemical Reactor

The cathodic reduction of oxygen to hydrogen peroxide, the current efficiency for the production of H₂O₂ and the oxidation of veratryl alcohol with an in situ generated hydrogen peroxide‐lignin peroxidase complex were studied in this paper. The complex was prepared by utilizing a novel preparation technique in an electrochemical reactor. The oxidation of veratryl alcohol (VA; 3,4‐dimethoxybenzyl alcohol) was carried out with or without lignin peroxidase under an electric field. The redox properties of veratryl alcohol on a carbon electrode in the presence of lignin peroxidase have been investigated using cyclic voltammetry. The kinetics of veratryl alcohol oxidation in an electrochemical reactor were compared to the oxidation when hydrogen peroxide was supplied externally. Further, the oxidation of veratryl alcohol by lignin peroxidase was optimized in terms of enzyme dosage, pH, and electrical potential. The novel electroenzymatic method was found to be effective using in situ generated hydrogen peroxide for the oxidation of veratryl alcohol by lignin peroxidase.     

The Synthesis of Dilnethyl α-Sorigenin

1,6,8-Trimethoxy-3-hydroxymethyl-2-naphthoic acid lactone (IV) was synthesized from benzoic acid in 21 steps. This lactone (IV) was completely identical with authentic dimethyl α-sorigenin, obtained by the methylation of natural α-sorigenin. Herewith the structure of α-sorigenin was confirmed to be 1,8-dihydroxy-6-methoxy-3-hydroxymethyl-2-naphthoic acid lactone (III).     

Anisaldehyde and Veratraldehyde Acting as Redox Cycling Agents for H2O2 Production by Pleurotus eryngii

The existence of a redox cycle leading to the production of hydrogen peroxide (H₂O₂) in the white rot fungus Pleurotus eryngii has been confirmed by incubations of 10-day-old mycelium with veratryl (3,4-dimethoxybenzyl) and anisyl (4-methoxybenzyl) compounds (alcohols, aldehydes, and acids). Veratraldehyde and anisaldehyde were reduced by aryl-alcohol dehydrogenase to their corresponding alcohols, which were oxidized by aryl-alcohol oxidase, producing H₂O₂. Veratric and anisic acids were incorporated into the cycle after their reduction, which was catalyzed by aryl-aldehyde dehydrogenase. With the use of different initial concentrations of either veratryl alcohol, veratraldehyde, or veratric acid (0.5 to 4.0 mM), around 94% of veratraldehyde and 3% of veratryl alcohol (compared with initial concentrations) and trace amounts of veratric acid were found when equilibrium between reductive and oxidative activities had been reached, regardless of the initial compound used. At concentrations higher than 1 mM, veratric acid was not transformed, and at 1.0 mM, it produced a negative effect on the activities of aryl-alcohol oxidase and both dehydrogenases. H₂O₂ levels were proportional to the initial concentrations of veratryl compounds (around 0.5%), and an equilibrium between aryl-alcohol oxidase and an unknown H₂O₂-reducing system kept these levels steady. On the other hand, the concomitant production of the three above-mentioned enzymes during the active growth phase of the fungus was demonstrated. Finally, the possibility that anisaldehyde is the metabolite produced by P. eryngii for the maintenance of this redox cycle is discussed.     

New antibiotic with typical plant anthraquinone structure obtained studying terrestrial and marine Streptomycetes

In our screening for new antibiotics from bacteria, the streptomycete isolate M097 from Jiaozhou Bay in China was found to produce aloesaponarin II (1a) and 1,6-dihydroxy-8-hydroxymethyl-anthraquinone (2). Similarly, a terrestrial streptomycete GW24/1694 produced 1a and its methyl ether, the new compound 1-hydroxy-6-methoxy-8-methyl-anthraquinone (1b). All structures were derived by spectrochemical analysis and by comparison with reference data. The results showed that the marine streptomycete isolate M097 and the terrestrial streptomycete GW24/1694 could be a promising material for studying the biosynthetic pathway of polyketides.     

Multiple Forms of Acid Phosphatase in Cotyledons of Vigna mungo Seedlings: Immunological Detection and Quantitation

Using a combination of column chromatography and gel electrophoresis,we have found that acid phosphatase in cotyledons of Vigna mungoseedlings is composed of at least six forms (Ia₁, Ia₂, Ib₁,Ib₂, IIa and IIb). We purified one of the major forms, Ia₁,as a polypeptide of 53 kDa. Using an antiserum raised againstthe enzyme Ia₁, we examined the immunological relationshipsbetween the multiple forms from cotyledons and the distributionof the enzyme in organs of maturing and germinating seeds. (Received December 25, 1989; Accepted July 11, 1990)     

Production of halogenated compounds byBjerkandera adusta

The white-rot fungusBjerkandera adusta produces volatile chlorinated phenyl compounds. The main compounds identified were 3-chloro-4-methoxybenzaldehyde (3-chloro-p-anisaldehyde), 3-chloro-4-methoxybenzyl alcohol (3-chloro-p-anisyl alcohol), 3,5-dichloro-4-methoxybenzaldehyde (3,5-dichloro-p-anisaldehyde), and 3,5-dichloro, 4-methoxybenzyl alcohol (3,5-dichloro-p-anisyl alcohol).p-Anisaldehyde, veratraldehyde and the corresponding alcohols,p-anisyl alcohol and veratryl alcohol were produced simultaneously. Even with a very low concentration of chloride in the medium (< 10^–5 m), chlorinated aromatic compounds were still observed. Addition of bromide to the culture medium led to the production of brominated compounds: 3-bromo-4-methoxybenzaldehyde, 3-bromo-4-methoxybenzyl alcohol, 3,5-dibromo-4-methoxybenzaldehyde and 3-bromo-5-chloro-4-methoxybenzaldehyde. These brominated compounds have not previously been reported as natural products. Although iodo-aromatic compounds were not produced by supplementation of the medium with iodide, isovanillin was found in the culture broth under these conditions. This compound may be formed by substitution of the iodine intermediate by a hydroxyl group on the third carbon of the ring. Diiodomethane or chloroiodomethane were also found. It is the first time that the production of halomethane has been related to the production of halogenated aromatic compounds. All the strains tested have these capabilities.     

Synthesis of 2-Hydroxy-3-p-tolyl-2-cyclopentenone and Its Application to Isolaurene

2-Hydroxy-3-p-tolyl-2-cyclopentenone (7), a potential starting material for synthesis of isolaurene (1), was prepared by photolysis of 2-p-toluenesulfonyloxy-2-cyclopentenone (6). Conversion of 7 into 2,5-dimethyl-2-p-tolyl-cyclopentanone (14) was carried out as follows. Methylation of 7 with methyl iodide gave 2-methoxy-3-p-tolyl-2-cyclopentanone (9), and the treatment of (9) with methyl magnesium iodide afforded 2-methyl-5-p-tolyl-2-cyclopentenone (11). This compound was allowed to react with methyl iodide in the presence of sodium methoxide to yield 2,5-dimethyl-2-p-tolyI-4-cyclopentenone (13), the hydrogenation of which over palladium charcoal gave 14.     

Synthesis of veratraldehyde from veratryl alcohol by lignin peroxidase with in situ electrogeneration of hydrogen peroxide in an electrochemical reactor

We report the synthesis of veratraldehyde from veratryl alcohol by Phanerochaete chrysosporium lignin peroxidase with in situ electrogeneration of hydrogen peroxide in an electroenzymatic reactor. The effects of operating parameters such as enzyme level, pH, and electrical potential on the efficiency of veratryl alcohol oxidation were investigated. Furthermore, we compared direct addition of hydrogen peroxide with electrogeneration of the material during enzymatic oxidation of veratryl alcohol. The electroenzymatic method using in situ-generated hydrogen peroxide was found to be effective for oxidation of veratryl alcohol by lignin peroxidase. The new method may be easily applied to biodegradation systems.     

Role of Veratryl Alcohol in Regulating Ligninase Activity in Phanerochaete chrysosporium

Ligninase activity in Phanerochaete chrysosporium is stimulated by incubating cultures with various substrates for the enzyme, including veratryl (3,4-dimethoxybenzyl) alcohol, which is a secondary metabolite of this fungus. This study was designed to provide insight into the mechanism involved in this stimulation. Ligninase activity increased 2 to 4 h after the addition of exogenous veratryl alcohol to ligninolytic cultures. This increase was prevented by inhibitors of protein synthesis. Analysis of the extracellular proteins by high-performance anion-exchange liquid chromatography revealed increases in the amounts of some, but not all, ligninase species. The normal rapid decrease in ligninase activity in aging cultures was not prevented or retarded by veratryl alcohol, indicating that veratryl alcohol does not increase ligninase activity by protecting extant enzyme. We conclude that veratryl alcohol probably functions via an induction type of mechanism, affecting only certain ligninase species. Results with an isolated lignin indicate that lignin (or its biodegradation products) functions in the same way that veratryl alcohol does.     

Characterization of four Rhodococcus alcohol dehydrogenase genes responsible for the oxidation of aromatic alcohols

Four genes were isolated and characterized for alcohol dehydrogenases (ADHs) catalyzing the oxidation of aromatic alcohols such as benzyl alcohol to their corresponding aldehydes, one from o-xylene-degrading Rhodococcus opacus TKN14 and the other three from n-alkane-degrading Rhodococcus erythropolis PR4. Various aromatic alcohols were bioconverted to their corresponding carboxylic acids using Escherichia coli cells expressing each of the four ADH genes together with an aromatic aldehyde dehydrogenase gene (phnN) from Sphingomonas sp. strain 14DN61. The ADH gene (designated adhA) from strain TKN14 had the ability to biotransform a wide variety of aromatic alcohols, i.e., 2-hydroxymethyl-6-methylnaphthalene, 2-hydroxymethylnaphthalene, xylene-α,α’-diol, 3-chlorobenzyl alcohol, and vanillyl alcohol, in addition to benzyl alcohol with or without a hydroxyl, methyl, or methoxy substitution. In contrast, the three ADH genes of strain PR4 (designated adhA, adhB, and adhC) exhibited lower ability to degrade these alcohols: these genes stimulated the conversion of the alcohol substrates by only threefold or less of the control value. One exception was the conversion of 3-methoxybenzyl alcohol, which was stimulated sevenfold by adhB. A phylogenetic analysis of the amino acid sequences of these four enzymes indicated that they differed from other Zn-dependent ADHs.The first two authors contributed equally to this work     

Transformation of 16-dehydroprogesterone and 17α-hydroxyprogesterone by Mucor piriformis

Mucor piriformis was used to study the mode of transformation of 16-dehydroprogesterone (I, pregna-4, 16-diene-3, 20-dione) and 17-hydroxyprogesterone (II, 17-hydroxypregn-4-ene-3, 20-dione). Biotransformation products formed from I were 14-hydroxypregna-4, 16-diene-3, 20-dione (Ia), 7, 14-dihydroxypregna-4, 16-diene-3, 20-dione (Ib), 3, 7, 14-trihydroxy-5-pregn-16-en-20-one (Ic), and 3, 7, 14-trihydroxy-5-pregn-16-en-20-one (Id). Metabolites Ic and Id appear to be hitherto unknown. Time-course studies suggested that the transformation is initiated by hydroxylation at the 14-position (Ia) followed by hydroxylation at the 7-position (Ib). Microsomes (105,000 g sediment) prepared from 16-dehydroprogesterone-induced cells hydroxylate I to its 14-hydroxy derivative (Ia) in the presence of NADPH. Incubation of Ia with the organism resulted in the formation of Ib, Ic and Id. Biotransformation products formed from compound II were 17, 20-dihydroxypregn-4-en-3-one (IIa), 7, 17-dihydroxypregn-4-ene-3, 20-dione (IIb), 6, 17, 20-trihydroxypregn-4-en-3-one (IIc) and 11, 17, 20-trihydroxypregn-4-en-3-one (IId). Time-course studies indicated that IIa is the initial product formed, which is further hydroxylated either at the 6 or 11 position. Incubation of IIa with the organism resulted in the formation of IIc and IId. Reduction of the 4-en-3-one system and 20-keto group has not been observed before in organisms of the order Mucorales. In addition, M. piriformis has been shown to carry out hydroxylation at the C-6, C-7, C-11 and C-14 positions in the steroid molecules tested.