Dicarboxylic acids and hydroxy fatty acids in different species of fungi

Secondary metabolites of fungi can be responsible for allergies; therefore, the identification of compounds produced by these organisms is very important. Fungi produce large amounts of secondary metabolites, which belong to groups of chemicals such as: dicarboxylic acids, hydroxy acids, alcohols, hydrocarbons, esters, fatty acids, sterols, amino acids and mycotoxins. The presence of all these compounds in human proximity contributes to many diseases. Therefore, the aim of the study was a qualitative and quantitative analysis of hydroxy and dicarboxylic acids produced by fungi occurring in student hostel in Poland, in the province of Pomerania. The following species of fungi were subjected to extraction: Aspergillus niger, Aspergillus fumigatus, Aspergillus candidus, Rhizopus sp., Geotrichum candidum, and Penicillium chrysogenum. A mixture of ethyl acetate and methanol was used for the extraction. The obtained extracts were further analyzed by gas chromatography mass spectrometry (GC–MS). In all samples of fungi, the presence of a total of 22 acids, including 13 dicarboxylic and 9 hydroxy acids, was confirmed. Most acids (17 different acids) were identified in A. fumigatus. Only 10 acids were identified in the mycelium of G. candidum and A. niger. Acids which were identified in all samples of the mycelium were 22-hydroxydocosanoic acid, 24-hydroxytetracosanoic acid and adipic acid. The most abundant compounds were 22-hydroxydocosanoic acid in A. fumigatus, A. candidus, Rhizopus sp., G. candidum and P. chrysogenum, and succinic acid in A. niger. More experiments are needed to understand the physiological role of hydroxy and dicarboxylic acids. We hope that our results are an important contribution to further studies on the human health.     

Investigation of Euphrasia rostkoviana Hayne Using GC–MS and LC–MS

Gas and liquid chromatography coupled with mass spectrometry was applied to solve difficulties and reinvestigate the serious matrix problems affecting analysis of the active compounds in Euphrasia rostkoviana Hayne. The main groups of compounds were obtained by extracting the herb stepwise with n-hexane, chloroform, ethyl acetate and methanol. Polyamide column chromatography facilitated further separation. Phenolic/flavonoid- and terpenoid-type molecules were studied by GC–MS, HPLC and LC–MS–MS. The β-sitosterol content of the herb was determined by gas chromatography with flame ionisation detection (GC-FID). Caffeic acid, chlorogenic acid, coumaric acid and flavonoid glycosides of apigenin, luteolin, rhamnetine (hexoside), kaempferol (both hexoside and rutinoside) and quercetin (rutinoside) were identified in the fractions of the methanolic extract.     

Kinetics of thermal decomposition of sulfur-containing amino acids

Rates of thermal decomposition of sulfur-containing amino acids such as D,L-methionine, L-cysteine, and L-cystine are studied. It is established that the amino acids decompose at 190–240°C to give the gaseous and liquid decomposition products in the polyphasic system formed. The rate of summary process is described by the first order kinetic equation up to 30–50% conversion. In spite of close values of the effective activation energies of thermal decomposition of D,L-methionine, L-cysteine, and L-cystine (195, 193, and 184 kJ mol^?1 respectively) the effective rate constants at one and the the same temperature differ by one or two orders of magnitude in the above-mentioned series. Sulfur-containing compounds prevail in the gaseous decomposition products, while in the liquid phase the nitrogen-containing ones are accumulated.     

Medium-Chain Fatty Acids (MCFA) Production Through Anaerobic Fermentation Using <Emphasis Type="Italic">Clostridium kluyveri</Emphasis>: Effect of Ethanol and Acetate

Medium-chain fatty acids (MCFA) are saturated monocarboxylic acids and can be used as antimicrobials, corrosion inhibitors, precursors in biodiesel, and bioplastic production. In the present study, MCFA production was evaluated with acetate and ethanol using the bacteria Clostridium kluyveri. Effects of substrate, electron donor, and methane inhibitor on MCFA production were evaluated. Bacteria successfully converted the ethanol and acetate to butyrate (C4), caproate (C6), and caprylate (C8) by chain elongation process. The highest concentrations of butyrate (4.6 g/l), caproate (3.2 g/l), and caprylate (0.5 g/l) were obtained under methane inhibition conditions than other conditions. The productions of butyrate and caproate were 1.6 and 1.48 times higher under methane inhibition conditions, respectively. Results denoted that the bacteria C. kluyveri can be used for conversion of acetate and ethanol into useful products like butyrate and caproate.     

Synthesis of amino acid conjugates of glycyrrhizic acid using <Emphasis Type="Italic">N</Emphasis>-hydroxyphthalimide and <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>'-dicyclohexylcarbodiimide

Synthesis of amino acid conjugates of glycyrrhizic acid with the use of N-hydroxyphthalimide, N,N'-dicyclohexylcarbodiimide, and tert-butyl esters of L-amino acids (valine, isoleucine, phenylalanine, and methionine) was performed followed by deprotection with trifluoroacetic acid. The target amino acid conjugates were isolated by column chromatography on silica gel in 40–45% yield. The structure of the prepared compounds was confirmed by IR and ¹³C NMR spectroscopy.     

Influence of solvents and novel extraction methods on bioactive compounds and antioxidant capacity of <Emphasis Type="Italic">Phyllanthus amarus</Emphasis>

Phyllanthus amarus (P. amarus) is a herbal plant used in the treatment of various diseases such as hepatitis, diabetes, and cancer. Efficiency of its bioactive compounds extraction and therefore the biological activity of the extracts are significantly influenced by both solvent character and extraction method. This study is aimed at the determination of the influence of six various solvents (water, acetonitrile, ethanol, methanol, ethyl acetate, and dichloromethane) and nine different extraction methods (conventional, ultrasound-assisted, microwave-assisted, and six novel methods) on the extraction efficiency and antioxidant capacity of P. amarus. The results indicated that water extracted the maximal amount of phenolics from P. amarus and had the highest antioxidant capacity, while microwave-assisted extraction provided the highest yields of phenolics and saponins, and the highest antioxidant capacity with the lowest energy consumption when compared to the other extraction methods. These findings implied that water and microwave-assisted extraction are recommended as the most effective solvent and method for the extraction of bioactive compounds from P. amarus for potential application in the pharmaceutical and nutraceutical industries.     

5-Trifluoromethylfuryl derivatives of phosphocarboxylic acids

Methods of synthesis of trifluoromethylfuryl derivatives of phosphonocarboxylic acids are studied. By addition of diethyl hydrogen phosphite to alkyl 3-(5-trifluoromethylfur-2-yl)acrylate under the conditions of the Pudovik reaction the corresponding derivative of 3-phosphonopropionic acid was prepared. Diethyl (5-trifluoromethylfur-2-yl)methanephosphonate in presence of potassium tert-butylate reacts with ethyl acrylate to form trifluoromethylfuryl derivative of 4-phosphonobutyric or 4-phosphonopimelic acid depending on the reaction conditions. In the products of reaction of the alkyl 3-(5-trifluoromethylfur-2-yl)-3-(diethoxyphosphoryl) propionate with ethyl acrylate in the presence of potassium tert-butylate formation of trifluoromethylfuryl derivative of the 3-phosphonoadipic acid is detected. 3-(5-Trifluoromethylfur-2-yl)-3-(diethoxyphosphoryl) propionic acid and its acid chloride are synthesized. The latter compound is used for acylation of glycine to form the corresponding N-acyl derivative. It is suggested that such compounds may be transported in the cell using usual channels of transportation of the amino acids and short peptides.     

Two directions of the reaction of 3,4-dihydroxy-6-oxoalka-2,4-dienoic acid esters with anthranilic acid hydrazide

Methyl 3,4-dihydroxy-6-oxoalka-2,4-dienoates reacted with anthranilic acid hydrazide to give methyl [5-alkyl-1-(2-aminobenzamido)-2-hydroxy-3-oxo-2,3-dihydro-1H-pyrrol-2-yl]acetates. The reaction of anthranilic acid hydrazide with ethyl 3,4-dihydroxy-6-oxohepta-2,4-dienoate afforded ethyl (2Z)-(3a-hydroxy-2-methyl-10-oxo-3,3a,5,10-tetrahydro-4H-pyrazolo[5,1-c][1,4]benzodiazepin-4-ylidene)acetate as solvate with one methanol molecule. The structure of the isolated compounds was determined on the basis of IR and NMR spectra and X-ray diffraction data.     

Synthesis of ethyl (2<Emphasis Type="Italic">E</Emphasis>,4<Emphasis Type="Italic">E</Emphasis>)- and (2<Emphasis Type="Italic">E</Emphasis>,4<Emphasis Type="Italic">Z</Emphasis>)-5-chloropenta-2,4-dienoates

An efficient synthetic approach to 2E,4E and 2E,4Z isomers of ethyl 5-chloropenta-2,4-dienoate has been developed on the basis of one-pot oxidation–olefination of readily accessible (E)- and (Z)-3-chloroprop-2-en-1-ols by the action of barium manganate and ethyl (triphenyl-λ⁵-phosphanylidene)acetate.     

Reduction and diazotization of ethyl 7-amino-3-<Emphasis Type="Italic">tert</Emphasis>-butyl-4-oxo-4,6-dihydropyrazolo[5,1-<Emphasis Type="Italic">c</Emphasis>][1,2,4]triazine-8-carboxylate

The ester group in ethyl 7-amino-3-tert-butyl-4-oxo-4,6-dihydropyrazolo[5,1-c][1,2,4]triazine-8-carboxylate was converted for the first time to methyl by the action of lithium tetrahydridoborate in the presence of boron trifluoride–diethyl ether complex. This reaction has almost no analogies among other classes of organic compounds. New difficultly accessible 7-chloro and 7-azido derivatives were synthesized via diazotization of the reduction product, and treatment of the latter with acetic anhydride afforded the exhaustively acetylated derivative. Diazotization of ethyl 7-amino-3-tert-butyl-4-oxo-4,6-dihydropyrazolo-[5,1-c][1,2,4]triazine-8-carboxylate, followed by reaction with sodium azide, gave the corresponding azide, and the product of azo coupling with ethyl acetoacetate failed to undergo intramolecular cyclization to tricyclic pyrazolo[3,2-c: 5,1-c′]bis[1,2,4]triazine system.     

Characterization of Truncated <Emphasis Type="Italic">dsz</Emphasis> Operon Responsible for Dibenzothiophene Biodesulfurization in <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. FUM94

Numerous desulfurizing bacteria from the Rhodococcus genus harbor conserved dsz genes responsible for the degradation of sulfur compounds through 4S pathway. This study describes a newly identified desulfurizing bacterium, Rhodococcus sp. FUM94, which unlike previously identified strains encodes a truncated dsz operon. DNA sequencing revealed a frameshift mutation in the dszA gene, which led to an alteration of 66 amino acids and deletion of other C-terminal 66 amino acids. The resulting DszA polypeptide was shorter than DszA in Rhodococcus sp. IGTS8 reference strain. Despite the truncation, desulfurizing activity of the operon was observed and attributed to the removal of an overlap of dszA and dszB genes, and lack of active site in the altered region. Desulfurization experiments resulted in specific production rate of 6.3 mmol 2-hydroxy biphenyl (kgDCW)^?1 h^?1 at 2 g l^?1 biocatalyst concentration and 68.8% biodesulfurization yield at 20 g l^?1 biocatalyst concentration, both at 271 μM dibenzothiophene concentration which is comparable to similar wild-type biocatalysts.     

Determination of astaxanthin and astaxanthin esters in the microalgae <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis> by LC-(APCI)MS and characterization of predominant carotenoid isomers by NMR spectroscopy

The oily product ZANTHIN® consists of natural astaxanthin, which is manufactured from the microalgae Haematococcus pluvialis by supercritical CO₂ extraction. An HPLC method was developed to separate all of the components of the complex astaxanthin extract using a C₃₀ column. The separation resulted in different isomers of astaxanthin accompanied by two other carotenoids. The main component consisted of astaxanthin singly esterified with several different fatty acids. C18:3, C18:2, C18:1 and C16:0 were identified as the most commonly occurring fatty acids. Doubly esterified astaxanthin was also found, although in lower concentrations compared to singly esterified astaxanthin. After performing a detailed fatty acid analysis by GC-MS, the peaks from the extract were assigned via HPLC-MS. A trans to cis transmutation of the all-trans compound was performed by thermal treatment in order to obtain an enrichment of cis isomers as the basis for unambiguous identification via NMR experiments. The all-trans as well as the 9- and 13-cis isomers of astaxanthin were characterized in detail by UV/Vis, ¹H, and ¹H,¹H COSY NMR spectroscopy.     

Quantum chemical studies of pyrimidin-4-ones Part 6. On peculiarities of the reactions with nitrating agents of some carboxylic acids of the thieno[2,3-<Emphasis Type="Italic">d</Emphasis>]pyrimidin-4-one series and their esters

Quantum chemical calculations of the energies, electronic structures, and molecular geometries of 5-methyl-, 2,5-dimethyl-, 3,5-dimethyl-, and 2,3,5-trimethyl-4-oxothieno[2,3-d]pyrimidine-6-carboxylic acids; 5-methyland 3,5-dimethyl-2,4-dioxothieno[2,3-d]pyrimidine6-carboxylic acids; 5-methyland 3,5-dimethyl-4-oxo-2-thiothieno[2,3-d]pyrimidine-6-carboxylic acids; and their ethyl esters were performed in terms of the HF and DFT (B3LYP) approximations in the 3-21G basis set. The reactions routes with nitrating agents were examined, and the factors governing the direction of the reactions, the probability of their occurrence were determined. Selective ipso-substitution of the 5-methyl group by the nitro group at the C(5) atom of the thiophene fragment is hindered for all compounds studied. The reasons for the unfavorable ipso-substitution were discussed taking into account effects of solvents and substituents in positions 2, 3, and 6 of 6-carboxyand 6-ethoxycarbonyl-substituted thieno[2,3-d]pyrimidin-4-ones.     

Effect of temperature on longevity of <Emphasis Type="Italic">Diaphorina citri</Emphasis> (Hemiptera: Liviidae) studied by microcalorimeter

The longevity of Asian citrus psyllid, Diaphorina citri, infected by an entomopathogenic fungus, Isaria fumosorosea, was studied by microcalorimetry. Experimental results of microcalorimetry suggest that in the process of metabolism of D. citri heat or heat flow declined exponentially. The metabolism of D. citri inhibited by increasing temperature, and the longevity of D. citri decreased gradually with respect to temperature. The longevity of D. citri almost reaches 500 h at 25 °C and D. citri died within 130 h at 41 °C under high-temperature exposure. The same phenomenon, decrease in longevity and metabolism inhibition, was observed under the low-temperature exposure. The supercooling point of insects can be derived from a cooling curve plotting the temperature versus time by using microcalorimetry. The heat released due to phase change can be determined by using microcalorimeter. Our results indicated that microcalorimetry can be used to measure the metabolism of herbivore insects.     

Improved Treatment and Utilization of Rice Straw by <Emphasis Type="Italic">Coprinopsis cinerea</Emphasis>

As one of the most abundant renewable resources, rice straw is an attractive lignocellulosic material for animal feeding or for the production of biochemical. An appropriate pre-treatment technique is essential for converting rice straw to rich fodder or biofuel. Based on previous work, Coprinopsis cinerea can grow on rice straw medium and therefore it is useful for the treatment of rice straw. However, little is known regarding its degradation systems and nutrition values. In this study, we firstly found that C. cinerea could grow rapidly on rice straw without any additives by the production of a series of enzymes (laccase, cellulase, and xylanase) and that the microstructure and contents of rice straw changed significantly after being treated by C. cinerea. We propose that a possible underlying mechanism exists in the degradation. Moreover, C. cinerea has a high nutrition value (23.5% crude protein and 22.2% total amino acids). Hence, fermented rice straw with mycelium could be a good animal feedstuff resource instead of expensive forage. The direct usage of C. cinerea treatment is expected to be a practical, cost-effective, and environmental-friendly approach for enhancing the nutritive value and digestibility of rice straw.     

Production and Biotechnological Application of Extracellular Alkalophilic Lipase from Marine Macroalga-Associated <Emphasis Type="Italic">Shewanella algae</Emphasis> to Produce Enriched C<Subscript>20-22</Subscript><Emphasis Type="Italic">n</Emphasis>-3 Polyunsaturated Fatty Acid Concentrate

An extracellular alkalophilic lipase was partially purified from heterotrophic Shewanella algae (KX 272637) associated with marine macroalgae Padina gymnospora. The enzyme possessed a molecular mass of 20 kD, and was purified 60-fold with a specific activity of 36.33 U/mg. The enzyme exhibited V_max and K_m of 1000 mM/mg/min and 157 mM, respectively, with an optimum activity at 55 °C and pH 10.0. The catalytic activity of the enzyme was improved by Ca²⁺ and Mg²⁺ ions, and the enzyme showed a good tolerance towards organic solvents, such as methanol, isopropanol, and ethanol. The purified lipase hydrolyzed the refined liver oil from leafscale gulper shark Centrophorus squamosus, yielding a total C_20-22 n-3 PUFA concentration of 34.99% with EPA + DHA accounting the major share (34% TFA), after 3 h of hydrolysis. This study recognized the industrial applicability of the thermostable and alkalophilic lipase from marine macroalga-associated bacterium Shewanella algae to produce enriched C_20-22 n-3 polyunsaturated fatty acid concentrate.     

Synthesis of (2<Emphasis Type="Italic">R</Emphasis>,4<Emphasis Type="Italic">R</Emphasis>)-2-alkyl-3-(2-mercaptobenzoyl)thiazolidine-4-carboxylic acids

Basing on natural amino acid L-cysteine, commercially available aliphatic aldehydes, 2-acetylsulfanylbenzoyl chloride and 2,2′-disulfandiyldibenzoyl dichloride a synthesis was developed of (2R,4R)-2-alkyl-3-(2-mercaptobenzoyl)thiazolidine-4-carboxylic acids, potential antihypertensive compounds, inhibitors of angiotensin transforming enzyme.     

Reaction of polychloroacetaldehyde arylsulfonylimines with 2-amino-6<Emphasis Type="Italic">H</Emphasis>-1,3-thiazine-6-thiones and 2-amino-4-phenyl-6<Emphasis Type="Italic">H</Emphasis>-1,3-thiazin-6-one

2-Amino-4-R-6H-1,3-thiazine-6-thiones and 2-amino-4-phenyl-6H-1,3-thiazin-6-one react with highly electrophilic N-arylsulfonylimines of chloral and phenyldichloroacetic aldehyde at the exocyclic amino group affording in good yields products of nucleophilic addition to the azomethine group of imines: N-[2-polychloro-1-(6-thioxo-4-R-6H-1,3-thiazin-2-ylamino)ethyl]- or -[2-polychloro-1-(6-oxo-4-phenyl-6H-1,3-thiazin-2-ylamino)ethyl]arenesulfonamides.     

Thermal Rearrangements of 3<Emphasis Type="Italic">H</Emphasis>- and 4<Emphasis Type="Italic">H</Emphasis>-Pyrazoles Prepared by Reactions of 9-Diazofluoren with Methyl Tetrolate and Methyl 3-Phenylpropiolate

9-Diazofluoren adds in Et₂O at 20°C to methyltetrolate in keeping with Auwers rule and nonregioselectively adds to methyl-3-phenylpropiolate with the formation of spirocyclic 3H-pyrazoles. The methyltetrolate adduct at boiling in toluene converts into methyl 3a-methyl-3aH-dibenzo[e,g]indazole-3-carboxylate, at 190°C in benzene, into methyl 3-methyl-2H-dibenzo[e,g]indazole-2-carboxylate, and at 160°C in methanol, into 3-methyl-2H-dibenzo[e,g]indazole. Auwers adduct of methyl 3-phenylpropiolate at boiling in benzene gives cyclopropene derivative and at boiling in methanol isomerizes into methyl 3a-phenyl-3aHdibenzo[e,g]indazole-3-carboxylate. Anti-Auwers adduct at boiling in benzene isomerizes into methyl 2-phenylpyrazolo[1,5-f]phenanthridine-3-carboxylate.