Production of chiral epoxides by an ethene-utilisingMicrococcus sp.

Summary An ethene-utilising bacterium was isolated in pure culture from soil and was tentatively identified as aMicrococcus sp. The organism accumulated epoxyalkanes (0.2–13 mM) from internal, terminal, cyclic and aryl-substituted olefins and exhibited a substrate specificity which was different from that expected on the basis of the chemical reactivity pattern in peracid epoxidations. Epoxyalkanes were hydrolysed at a much slower rate than the epoxidation step which allowed them to accumulate. Ethene-grown cells catalysed the stereospecific formation of R-1,2-epoxypropane (enantiomeric excess: e.e.=96%), R-1,2-epoxybutane (e.e.=94%) andtrans-(2R,3R)-epoxybutane (e.e.=84%). An ethene monooxygenase was implicated in the production of chiral epoxides in cell-free extracts of the bacterium. The (2S,3S)-enantiomer of racemictrans-2,3-epoxybutane was stereoselectively hydrolysed to completion resulting in an enrichment in the (2R,3R)-enantiomer. Further hydrolysis of 1,2-epoxyalkanes (C₃-C₄), however, occurred via complete destruction of both stereoisomers.     

Oxidation of primary aliphatic alcohols by Acetobacterium carbinolicum sp. nov., a homoacetogenic anaerobe

Four strains of new homoacetogenic bacteria were enriched and isolated from freshwater sediments and sludge with ethanol, propanol, 1,2-propanediol, or 1,2-butanediol as substrates. All strains were Gram-positive nonsporeforming rods and grew well in carbonate-buffered defined media under obligately anaerobic conditions. Optimal growth occurred at 27° C around pH 7.0. H₂/CO₂, primary aliphatic alcohols C₃–C₅, glucose, fructose, lactate, pyruvate, ethylene glycol, 1,2-propanediol, 2,3-butanediol, acetoin, glycerol, and methyl groups of methoxylated benzoate derivates and betaine were fermented to acetate or, in case of primary alcohols C₃–C₅ and 1,2-propanediol, to acetate and the respective fatty acid. In coculture with methanogens methane was formed, probably due to interspecies hydrogen transfer. Strain WoProp 1 is described as a new species, Acetobacterium carbinolicum. It had a DNA base composition of 38.5±1.0% guanine plus cytosine, and contained murein of crosslinkage type B similar to A. woodii.     

Kinetic resolution of ketoprofen ester catalyzed by lipase from a mutant of CBS 5791

A biotransformation process was developed for the production of (S)-ketoprofen by enantioseletive hydrolysis of racemic ketoprofen ester using the mutant Trichosporon laibacchii strain CBS 5791. A satisfactory result was obtained, in which the E was 82.5, with an ee of 0.94 and a conversion of 0.47 under the optimum hydrolysis conditions [E is enantiomeric ratio, E=ln[1–X(1+ee)]/ln[1–X(1–ee)]; ee is enantiomeric excess, ee=(C_S–C_R)/(C_S+C_R): temperature of hydrolysis was 23°C]. The medium used in biotransformation was a mixture of growth broth and biotransformation broth at a ratio of 1:9, the concentration of Tween 80 was 15 g/l, the time of hydrolysis, 72 h. These results are promising for further scale-up. Tween 80 significantly improved lipase enantioselectivity and activity at the optimum concentration.     

Synthesis of new 4-(E)-alkenylpyrrolo[1,2-a]quinoxalines as antileishmanial agents by Suzuki-Miyaura cross-coupling reactions

A series of new 4-(E)-alkenylpyrrolo[1,2-a]quinoxaline derivatives, structural analogues of alkaloid chimanine B, was synthesized in good yields using efficient palladium(0)-catalyzed Suzuki-Miyaura cross-coupling reactions. These new compounds were tested for in vitro antiparasitic activity upon Leishmania amazonensis and Leishmania infantum strains. Biological results showed activity against the promastigote forms of L. amazonensis and L. infantum with IC₅₀ ranging from 0.5 to 7 μM. From a Structure-Activity Relationships point of view, these pharmacological results mainly enlightened the importance of the 4-lateral C₆, C₇ or C₈ α-unsaturated trans-alkenyl chain of unsubstituted pyrrolo[1,2-a]quinoxaline moiety.     

Substrate Specificity and Mode of Action of the Lipase Produced by Pseudomonas fragi22.39 B

The lipase purified from Pseudomonas fragi 22.39 B hydrolyzed not only triglycerides but also synthetic esters such as Tween, Span and methyl oleate. Of the saturated monoacid triglycerides tested, tributyrin was hydrolyzed most quickly. The lipase did not produce 1,3-diolein as a hydrolysis product from triolein. The addition of the Ca²⁺ ion to the reaction mixture promoted the hydrolysis rate for triglycerides and monoesters with longer-chain fatty acids (C₁₄, C₁₆, C₁₈). The enzyme could hydrolyze various kinds of natural fats and oils, and the extent their hydrolysis reached above 90%.     

Production of 1,2-epoxyalkanes from 1-alkenes by Nocardia corallina B-276

Summary A gaseous hydrocarbon-assimilating microorganism, Nocardia corallina, grew on 1-alkenes (C₃, C₄ and C₁₃ to C₁₈) and produced corresponding 1,2-epoxyalkanes. One of the products, 1,2-epoxytetradecane, had a positive rotation [] _D ²⁰ =+8.6°. The concentration of propyleneoxide reached 0.6 g/l after 5 days of cultivation. Accumulation of 1,2-epoxytetradecane continued till the end of cultivation when the pH was not controlled, though production stopped in a 24 h cultivation when the pH was controlled at an optimum pH for growth. Propylene-grown cells produced 1,2-epoxytetradecane from 1-tetradecene and degraded tetradecane. In the presence of chloramphenicol, propylene-grown cells did not degraded tetradecane though 1,2-epoxytetradecane was produced from 1-tetradecene. A spontaneous mutant, which neither grew on propylene nor produced propyleneoxide, was isolated. This strain grew on tetradecane and 1-tetradecene but produced only a trace amount of 1,2-epoxytetradecane from 1-tetradecene.     

Improved gas chromatographic assay for the simultaneous determination of nitroglycerin and its mono- and dinitrate metabolites

A sensitive, specific capillary gas chromatographic—electron-capture detection method for the simultaneous determination of nitroglycerin (GTN), 1,2- and 1,3-glyceryl dinitrate (1,2-GDN and 1,3-GDN, respectively) and 1- and 2-glyceryl mononitrate (1-GMN and 2-GMN, respectively) is reported. The minimum quantifiable concentration for GTN, GDNs and GMNs is 0.4 ng/ml in plasma, with extraction recoveries for GMNs>76% and for GTN and the GDNs>95%. Over the full range of quantifiable concentrations the inter-run assay precision and accuracy were less than 13 and 11%, respectively, for all five nitrates. Similar intra-run assay precision and accuracy values were found. The method was employed in the preliminary in vitro examination of GTN, GDN and GMN kinetics in human blood. Following addition of GTN to human blood, the ratio of 1,2-GDN to 1,3-GDN maximum concentrations (C_max) was ca. 7:1, reflecting preferential denitration of the GTN molecule at the primary positions, while the C_max ratio for 2-GMN to 1-GMN in this system was ca. 6:1, representing a highly selective if not specific primary denitration of the 1,2-GDN molecule. Following the intravenous administration of 1,2-GDN to five healthy male volunteers, 2-GMN/1-GMN C_max ratios averaged 8.8:1, representing a highly selective but not specific formation of 2-GMN from the 1,2-GDN molecule. The assay will find utility in in vitro studies attempting to address the molecular pharmacology of GTN and its metabolites, and in in vivo clinical pharmacology studies attempting to address the relationship between pharmacokinetics and pharmacodynamics of GTN and its metabolites.     

A computational investigation of sulfur-containing heterocyclic components from the anal sac secretions of Mustela species

A computational investigation of the sulfur-containing heterocyclic components (substituted thietanes and 1,2-dithiolanes) of Mustela anal sac secretions has been carried out. A cluster analysis of the chemical compositions of Mustela anal sac volatiles reveals little similarity with established phylogenetic relationships between members of the genus. Ab initio calculations [MP2/6–311++G(2df,2p)//B3LYP/6–311++G**] show the lowest-energy C₅H₁₀S isomeric thietane to be 2,2-dimethylthietane, which is also the most abundant of the Mustela thietanes. Similarly, 3,3-dimethyl-1,2-dithiolane is the lowest-energy C₅H₁₀S₂ compound. 2-n-Propylthietane is the highest-energy C₆H₁₂S compound, but the most abundant Mustela C₆H₁₂S compound produced, whereas cis-2-ethyl-4-methylthietane, the lowest-energy C₆H₁₂S thietane, has never been observed in Mustela anal sac secretions. A molecular docking analysis of the Mustela sulfur-containing heterocycles into both porcine and bovine odorant binding proteins reveals the interactions of the docked ligands with the proteins to be largely hydrophobic, and have binding energies generally lower than typical odorant molecules such as linalool or eugenol. Figure Mustela anal sac volatile components, 2,2-dimethylthietane and cis-3,4-dimethyl–1,2-dithiolane.     

Membrane restructuring by phospholipase A2 is regulated by the presence of lipid domains

Secretory phospholipase A₂ (sPLA₂) catalyzes the hydrolysis of glycerophospholipids. This enzyme is sensitive to membrane structure, and its activity has been shown to increase in the presence of liquid-crystalline/gel (L_α/L_β) lipid domains. In this work, we explore whether lipid domains can also direct the activity of the enzyme by inducing hydrolysis of certain lipid components due to preferential activity of the enzyme toward lipid domains susceptible to sPLA₂. Specifically, we show that the presence of L_α/L_β and L_α/P_β′ phase coexistence in a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2 distearoyl-sn-glycero-3-phosphocholine (DSPC) system results in the preferential hydrolysis of the shorter-chained lipid component in the mixture, leading to an enrichment in the longer-chained component. The restructuring process is monitored by atomic force microscopy on supported single and double bilayers formed by vesicle fusion. We observe that during preferential hydrolysis of the DMPC-rich L_α regions, the L_β and P_β′ regions grow and reseal, maintaining membrane integrity. This result indicates that a sharp reorganization of the membrane structure can occur during sPLA₂ hydrolysis without necessarily destroying the membrane. We confirm by high-performance liquid chromatography the preferential hydrolysis of DMPC within the phase coexistence region of the DMPC/DSPC phase diagram, showing that this preferential hydrolysis is accentuated close to the solidus phase boundary. Differential scanning calorimetry results show that this preferential hydrolysis in the presence of lipid domains leads to a membrane system with a higher-temperature melting profile due to enrichment in DSPC. Together, these results show that the presence of lipid domains can induce specificity in the hydrolytic activity of the enzyme, resulting in marked differences in the physical properties of the membrane end-product.     

Adsorption and recovery of cellulases during hydrolysis of newspaper

The adsorption of cellulases from Trichoderma viride was studied during the hydrolysis of newspaper. By measuring individual enzyme activities it was found that in the early stage of hydrolysis enzyme components showing C_xA; were adsorbed preferentially to those showing C₁A; afterwards ths situation was inverted. Electrophoretic resolution of proteins in hydrolysates showed a continuous decrease of enzyme proteins in solution, and furthermore suggested that the enzymes once adsorbed remained immobilized on the substrate (even after extensive digestion). Experiments to recover the enzyme that had remained in solution after typical hydrolysis showed a potential saving of up to 40%.     

Substrate specificity and stereospecificity of limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis DCL14; an enzyme showing sequential and enantioconvergent substrate conversion

Limonene-1,2-epoxide hydrolase (LEH) from Rhodococcus erythropolis DCL14, an enzyme involved in the limonene degradation pathway of this microlorganism, has a narrow substrate specificity. Of the compounds tested, the natural substrate, limonene-1,2-epoxide, and several alicyclic and 2-methyl-1,2-epoxides (e.g. 1-methylcyclohexene oxide and indene oxide), were substrates for the enzyme. When LEH was incubated with a diastereomeric mixture of limonene-1,2-epoxide, the sequential hydrolysis of first the (1R,2S)- and then the (1S,2R)-isomer was observed. The hydrolysis of (4R)- and (4S)-limonene-1,2-epoxide resulted in, respectively, (1S,2S,4R)- and (1R,2R,4S)-limonene-1,2-diol as the sole product with a diastereomeric excess of over 98%. With all other substrates, LEH showed moderate to low enantioselectivities (E ratios between 34 and 3).     

A Comparison of Growth Inhibition of Tetrahymena furgasoni by C19 and C21 Steroids

The growth inhibition of Tetrahymena furgasoni (once known as “T. pyriformis W”) by C₁₉ and C₂₁ steroids of similar structure was measured by determining cell population at 24 h and 48 h following addition of the steroid. A cis-fusion of the A/B rings junction, unsaturation at C-1,2, or C-4,5 and carbonyl substitution all enhanced inhibition, whereas the presence of two hydroxyl groups decreased inhibition. The results indicated that the transformation of C₁₉ and C₂₁ steroids by this protozoon may be part of a detoxication mechanism.     

Structure Determination of Fatty Acids from Tunicamycin Complex

The structures of ten fatty acids, which were obtained by the hydrolysis of tunicamycin complex, were determined. GLC-mass, ¹H NMR and IR spectra showed that the major acids were trans-α, β-unsaturated iso acids with the formula C₁₄H₂₈O₂, C₁₆H₂₈O₂, C₁₆H₃₀O₂ and C₁₇H₃₂O₂. The minor acids were α, β-unsaturated normal acids and saturated normal and iso acids.     

Phospholipases. I. Effect ofn-alkanols on the rate of enzymatic hydrolysis of egg phosphatidylcholine

Summary The rate of hydrolysis of unsonicated liposomes of egg lecithin by phospholipase A (from bee venom and Russell viper venom) and phospholipase C (fromBacillus cereus andClostridium welchii) is markedly dependent on the nature and concentration of a variety of added alcohols. Typical plots of rate against alcohol concentration are bell-shaped. The maximum rate and the alcohol concentration at which it is achieved are alcohol-specific. In a homologous series ofn-alkanols, the maximal rates increase and the optimal concentrations decrease as the chain length is increased from C₄ to C₈. For longer alcohols (C₉ to C₁₂), progressively higher concentrations are required to elicit maximal activation. The optimal activating concentrationsC for C₄ to C₈ n-alkanols obey the relationshipp C=a logP _octanol+constant [cf. Hansch & Dunn,J. Pharm. Sci. 61:1 (1972)], suggesting that the alcohol-activating effect is a consequence of their incorporation into the liposomes with resultant modification of liposomal structure.     

Synthesis of a 6-c-nitro-d-glucopyranose derivative having phosphorus in the hemiacetal ring

5,6-Dideoxy-6-C-nitro-5-(phenylphosphino)-d-glucopyranose was prepared by addition of phenylphosphine to 3-O-acetyl-5,6-dideoxy-1,2-O-isopropylidene-6-C-nitro-α-d-xylo-hex-5-enofuranose, followed by hydrolysis of the resulting 3-O-acetyl-5,6-dideoxy-1,2-O-isopropylidene-6-C-nitro-5-(phenylphosphino)-d-glucofuranose (10). Acetylation of 10 gave the crystalline 1,2,3,4-tetraacetate (16). 5,6-Dideoxy-6-C-nitro-5-(phenylphosphinyl)-d-glucopyranose (15) was obtained by oxidation of 10, and hydrolysis of the resulting 5-phenylphosphinyl compound. Acetylation of 15 gave the 1,2,3,4-tetraacetate (17). Although the n.m.r. spectrum of 17 was complex, the n.m.r. spectrum of 16 was rather simple. The n.m.r. data showed that 16 is the α anomer in the ⁴C₁(d) conformation.     

Enrichment of very-long-chain mono-unsaturated fatty acids by lipase-catalysed hydrolysis and transesterification

Partial hydrolysis of triacylglycerols of high-erucic-acid seed oils from white mustard (Sinapis alba), oriental mustard (Brassica juncea) and honesty (Lunaria annua), catalysed by lipases from Candida cylindracea and Geotrichum candidum, leads to enrichment of erucic acid and other very-long-chain mono-unsaturated fatty acids (VLCMFA) in the acylglycerols (mono-, di- and triacylglycerol) while the C₁₈ fatty acids (oleic, linoleic and linolenic) are enriched in the fatty acid fraction. Partial hydrolysis of the high-erucic-acid triacylglycerols, catalysed by lipases from porcine pancreas, Chromobacterium viscosum, Rhizopus arrhizus and Rhizomucor miehei yields fatty acids with substantially higher levels of VLCMFA, as compared to the starting material, while the C₁₈ fatty acids are enriched in the acylglycerol fraction. Lipases from Penicillium sp. and Candida antarctica are ineffective for the fractionation of either group of fatty acids. Transesterification of the high-erucic-acid triacylglycerols with ethyl, propyl or butyl acetate or with n-butanol, catalysed by the lipase from R. miehei, leads to enrichment of VLCMFA in the alkyl (ethyl, propyl or butyl) esters, whereas the C₁₈ fatty acids are enriched in the acetylacylglycerols and acylglycerols.     

Enantioselective syntheses of isotopically labelledα-amino acids Preparation of specifically13C-labelled L-lysines

Summary [2-¹³C]-L-lysine, [3,4-¹³C₂]-L-lysine and [5,6-¹³C₂]-L-lysine are prepared from simple, commercially available, highly enriched starting materials as [2-¹³C]-glycine, ethyl [1,2-¹³C₂]-bromo acetate, and [1,2-¹³C₂]-acetonitrile. The introduction of the chiral center is based on a general method starting from the bis-lactim ether of cyclo-(D-Val-Gly). The synthesis of (2R)-[5-¹³C]-3,6-diethoxy-2,5-dihydro-2-isopropylpyrazine is described. The availability of our method for the preparation of specifically enriched bis-lactim ethers allows the synthesis of a great variety of site specific isotopically labelled (L- and D-)-amino acids. Moreover, intermediate 4-[(2R,5S)-3,6-diethoxy-2,5-dihydro-2-isopropyl-5-pyrazinyl]butyronitrile is a valuable precursor in the synthesis of L--aminoadipic acid. The synthetic scheme in this publication makes both L-lysine and L--aminoadipic acid¹³C- or¹⁵N-labelled at any position, easily available. The isotopomers of lysine are obtained on a preparative scale in good yields, with 99%¹³C and high enantiomeric purity (>97% e.e.). Three isotopomers are characterized using various spectroscopic techniques,e.g.,¹H NMR,¹³C NMR and Mass spectrometry.     

PHOSPHOLIPID METABOLISM IN SUBACUTE SCLEROSING PANENCEPHALITIS : ACTIVITY OF BRAIN PHOSPHOLIPASE A2 TOWARDS SPECIFICALLY LABELLED 1,2-DIACYL-, 1-ALK-1''-ENYL-2-ACYL- AND 1-ALKYL-2-ACYL-sn-GLYCERO-3-PHOSPHORYLCHOLINE

—1,2-Diacyl-, 1-alk-1′-eny1-2-acyl- and 1-alky1-2-acyl-sn-glycero-3-phosphorylcholine specifically labelled with different fatty acids at the 2 position, were prepared enzymically using the acyltransferase system of rabbit sarcoplasmic reticulum. The substrates were submitted to hydrolysis by phospholipase A₂ (phospholipid acyl-hydrolase, EC 3.1.1.4) obtained from normal and brain tissue affected with subacute sclerosing panencephalitis. In the diseased tissue an increase of phospholipase A₂ activity ranging from 46 to 54% could be observed in comparison to the control brain for all substrates investigated. Among the investigated substrates phospholipase A₂ had the highest affinity for the 1,2-diacylcompound, whereas alkenylacyl- and alkylacyl-sn-glycero-3-phosphorylcholine were cleaved at almost similar rates. The hydrolysis rate of choline-plasmalogen and the corresponding diacyl compound by the enzyme was greatly influenced by the fatty acid moiety located at the 2 position of the substrates.     

Effects of solvents on the production of epoxides by Nocardia corallina B-276

Summary Glucose-grown cells of Nocardia corallina B-276 produced epoxides from C₂ to C₁₈ 1-alkenes and styrene. Production of epoxides from C₆-C₁₀ 1-alkenes and styrene was enhanced by the addition of n-hexadecane as a solvent to the reaction system, while addition of n-hexadecane lecreased the rate of epoxidation of longer chain length 1-alkenes. n-Hexadecane was found to reduce substrate inhibition by styrene and product nhibition by styreneoxide and 1,2-epoxyalkanes. a-Alkanes, 1-alkenes, alkylbenzenes, halogenated akanes and a diester of a dibasic acid could be sed as solvents.     

Bacterial metabolism of side chain fluorinated aromatics: cometabolism of 4-trifluoromethyl(TFM)-benzoate by 4-isopropylbenzoate grown Pseudomonas putida JT strains

Enzymes of the p-cymene pathway in Pseudomonas putida strains cometabolized the intermediate analogue 4-trifluoromethyl(TFM)benzoate. Three products, 4-TFM-2,3-dihydro-2,3-dihydroxybenzoate, 4-TFM-2,3-dihydroxy-benzoate and 2-hydroxy-6-oxo-7,7,7-trifluorohepta-2,4-dienoate (7-TFHOD) were identified chemically and by spectroscopic proterties.Certain TFM-substituted analogue metabolites of the p-cymene pathway were transformed at drastically reduced rates.Hammett type analysis of ring cleavage reactions of 4-substituted 2,3-dihydroxybenzoates revealed the negative inductive and especially mesomeric effect of substituents to be rate determining. Whereas decarboxylation of 3-carboxy-7-TFHOD was not affected by fluorine substitution the subsequent hydrolysis of 7-TFHOD proceeded very slowly. The negative inductive effect of the TFM-group probably inhibited heterolysis of the carbon bond between C₅ and C₆ of 7-TFHOD.Abbreviations DHB 1,2-Dihydroxy-2-hydrobenzoate - DHC 2,3-Dihydro-2,3-dihydroxybenzoate, this compound was termed DHC simply to distinguish it from the similar 1,2-dihydroxy-2-hydrobenzoate (DHB) as described in the preceeding paper (Engesser et al. 1988) - HMS 2-Hydroxymuconic semialdehyde - HOD 2-Hydroxy-6-oxohepta-2,4-dienoate - 7-TFHOD 2-Hydroxy-6-oxo-7,7,7-trifluorohepta-2,4-dienoate - TFM Trifluoromethyl This work was supported, in part, by the Gesellschaft für Strahlen- und Umweltforschung, Neuherberg/München, FRG