Ergosterol peroxides as phospholipase A2 inhibitors from the fungus Lactarius hatsudake

Four ergosterol derivatives (1–4) have been isolated for the first time from the fruiting bodies of a basidiomycete fungus, Lactarius hatsudake, through activity-guided fractionation. Their structures were determined, using spectroscopic analysis, as: (22E,24R)-ergosta-5,7,22-dien-3β-ol (ergosterol, 1); 5,8-epidioxy-(22E,24R)-ergosta-6,22-dien-3β-ol (ergosterol peroxide, 2); 5,8-epidioxy-(24S)-ergosta-6-en-3β-ol (3); and (22E,24R)-ergosta-7,22-dien-3β,5,6β-triol (cerevisterol, 4). Compounds 2 and 3 showed selective inhibitory activity against Crotalus adamenteus venom phospholipase A₂ (PLA₂) enzyme, but not against Apis mellifcra bee venom PLA₂. The antiphospholipase A₂ activity of compounds 2 and 3 are reported here for the first time.     

Design of phosphonium ionic liquids for lipase-catalyzed transesterification

Ionic liquids are now recognized as solvents for use in lipase-catalyzed reactions; however, there still remains a serious drawback in that the rate of reaction in an ionic liquid is slower than that in a conventional organic solvent. To overcome this problem, attempts have been made to evolve phosphonium ionic liquids appropriate for lipase-catalyzed reaction; several types of phosphonium salts have been prepared and their capability evaluated for use as solvent for the lipase-catalyzed reaction. Very rapid lipase PS-catalyzed transesterification of secondary alcohols was obtained when 2-methoxyethyl(tri-n-butyl)phosphonium bis(trifluoromethanesulfonyl)imide ([MEBu₃P][NTf₂]) was used as solvent, affording the first example of a reaction rate superior to that in diisopropyl ether.     

Lipase-catalyzed transesterification in several reaction systems: An application of room temperature lonic liquids for bi-phasic production ofn-butyl acetate

Organic solvents are widely used in biotransformation systems. There are many efforts to reduce the consumption of organic solvents because of their toxicity to the environment and human health. In recent years, several groups have started to explore novel organic solvents called room temperature ionic liquids in order to substitute conventional organic solvents. In this work, lipase-catalyzed transesterification in several uni-and bi-phasic systems was studied. Two representative hydrophobic ionic liquids based on 1-butyl-3-methylimidazolum coupled with hexafluorophosphate ([BMIM][PF₆]) and bis[(trifluoromethylsulfonyl) imide] ([BMIM] [Tf₂N]) were employed as reaction media for the transesterification ofn-butanol. The commercial lipase, Novozym 435, was used for the transesterification reaction with vinyl acetate as an acyl donor, The conversion yield was increased around 10% in a water/[BMIM][Tf₂N], bi-phasic system compared with that in a water/hexane system. A higher distribution of substrates into the water phase is believed to enhance the conversion yield in a water/[BMIM][Tf₂N] system. Partion coefficients of the substrates in the water/[BMIM][Tf₂N] bi-phasic system were higher than three times that found in the water/hexane system, while n-butyl acetate showed a similar distribution in both systems. Thus, RTILs appear to be a promising substitute of organic solvents in some biotransformation systems.     

A simple method to determine the enantiomeric ratio in enantioselective biocatalysis

The enantiomeric ratio (E) is commonly used to characterize the enantioselectivity in enzyme-catalyzed kinetic resolution. In this paper this parameter is directly derived from the enantiomeric excess of substrate and product. This is formally more correct than using Chen's equation after calculating the degree of conversion from both ee values using the relation of Sih and Wu. New expressions and useful graphs have been generated for reversible and irreversible uni-uni reactions. The theoretical predictions have been verified experimentally for various reactions. Values for E and the thermodynamic equilibrium constant,K_EQ, were obtained for a ( -dehalogenase-catalyzed dehalogenation, a hydrolysis reaction by porcine pancreatic lipase, and for C. Cylindracea lipase-catalyzed esterification and transesterification. In view of the current developments in the field of chiral analysis, this method is an easily available tool in the quantitative treatment of enzyme-catalyzed resolution of enantiomers.     

Resolution of 2-phenoxy-1-propanols through the enantioselective acylation mediated by Achromobacter sp. lipase as biocatalyst

Enantioselective acylation employing vinyl alkanoates as acyl donors was exploited for the resolution of 2-(substituted phenoxy)-1-propanols carrying different substituents on the benzene ring using Achromobacter sp. lipase. These primary alcohols with an oxygen atom at the stereocenter, were resolved with moderate to good enantioselectivity, based on the enantiomeric ratio E (up to 27), through acylation with vinyl butanoate in diisopropyl ether, after the examination of potential factors affecting the reaction such as organic solvents and acyl donors. Using this procedure, enantiomerically enriched (R)-2-(4-chlorophenoxy)-1-propanol was prepared in 97% e.e. and 33% yield in a gram-scale reaction.     

Copper(II) complexes with (2-hydroxybenzyl-2-pyridylmethyl)amine–Hbpa: syntheses, characterization and crystal structures of the ligand and [Cu(II)(Hbpa)2](ClO4)2·2H2O

Copper(II) complexes were synthesized and characterized by means of elemental analysis, IR and visible spectroscopies, EPR and electrochemistry, as well as X-ray structure crystallography. The group consists of discrete mononuclear units with the general formula [Cu(II)(Hbpa)₂](A)₂·nH₂O, where Hbpa=(2-hydroxybenzyl-2-pyridylmethyl)amine and A=ClO₄ ⁻, n=2 (1), CH₃COO⁻, n=3 (2), NO₃ ⁻, n=2 (3) and SO₄ ²⁻, n=3 (4). The structures of the ligand Hbpa and complex 1 have been determined by X-ray crystallography. Complexes 1–4 have had their UV–Vis spectra measured in both MeCN and DMF. It was observed that the compounds interact with basic solvents, such that molecules coordinate to the metal in axial positions in which phenol oxygen atoms are coordinated in the protonated forms. The values were all less than 1000 M⁻¹ cm⁻¹. EPR measurements on powdered samples of 1–3 gave g/A values between 105 and 135 cm⁻¹, typical for square planar coordination environments. Complex 4·3H₂O exhibits a behaviour typical for tetrahedral coordination. The electrochemical behaviour for complexes 1 and 2 was studied showing irreversible redox waves for both compounds.     

Volatiles released from oak, a host tree for the bark beetle Scolytus intricatus

Volatile compounds emitted in different phases of oak (Quercus robur) development (bark, unopened buds, young developing leaves, and blossoms) were analyzed with the aim of finding possible host-plant attractants for the European oak bark beetle, Scolytus intricatus. Complex mixtures of aliphatic, aromatic, and terpenoid compounds were identified in the samples. (E)-2-Hexenal and hexanal dominated in samples of bark. In buds, (Z)-3-hexenyl acetate formed a substantial part of the mixture. In both leaves and blossoms (E,E)--farnesene was the main component.

Volatiles released from oak twigs and branches during both the maturation feeding and construction of maternal galleries by Scolytus intricatus were also analyzed. Most compounds found in the samples from females’ and males’ maturation feeding were identical. High contents of anisole, (E)-β-ocimene, -copaene, one unidentified sesquiterpenic hydrocarbon C₁₅H₂₄ and β-caryophyllene were found in both samples of twigs attacked by beetles. During the construction of maternal galleries by bark beetles in oak logs, monoterpene hydrocarbons such as p-cymene, (E)-β-ocimene, and γ-terpinene, and sesquiterpenes -copaene and β-caryophyllene were released in large quantities. No new compound appeared when males were added to the log with feeding females.     

Increased enantioselectivity in the enzymatic hydrolysis of amino acid esters in the ionic liquid 1-butyl-3-methyl-imidazolium tetrafluoroborate

The initial rate and enantioselectivity of enzymatic asymmetric hydrolysis of amino acid esters were examined in methylimidazolium-based ionic liquids with anions including tetrafluoroborate, chloride, bromide and bisulfate and in typical organic solvents. Papain displayed much higher enantioselectivity but lower activity in phosphate buffer solution of 1-butyl-3-methylimidazolium tetrafluoroborate BMIM·BF₄ than in other media tested (i.e. E=100, V ₀=0.21 mM min^-1 in BMIM·BF₄, E=2, V ₀=0.43 mM min^-1 in phosphate buffer, E=14-92, V ₀=0.22-0.25 mM min^-1 in organic solvents for D,L-phenylglycine methyl ester). The influence of BMIM·BF₄ on enzyme activity and enantioselectivity also varied with the substrate and the enzyme used. All of the enzymes assayed showed no activity or low enantioselectivity in the ILs with anions including chloride, bromide and bisulfate.     

Enzymatic resolution of 2-aryloxy-1-propanols via lipase-catalyzed enantioselective acylation using acid anhydrides as acyl donors

Pseudomonas sp. lipase-catalyzed enantioselective acylation procedure using acid anhydrides as acyl donors was exploited for the resolution of 2-aryloxy-1-propanols carrying different substituents on the benzene ring. These primary alcohols, which belong to primary alcohols with an oxygen atom at the stereocenter, were resolved generally with moderate to good enantioselectivity (E of up to 55) through the acylation with hexanoic anhydride in diisopropyl ether at 25 °C in a short reaction time. With the alcohol substrate, which gave a low enantioselectivity in the acylation at ordinary temperature, the selectivity proved to be enhanced by conducting the reaction at low temperature (−10 °C). By this acylation procedure employing the acid anhydride, enantiomerically pure (R)-2-phenoxy-1-propanol was prepared in a gram-scale reaction.     

An Insight into the Active Site of Pseudomonas Fluorecens (P. cepacia) Lipase to Define the Stereochemical Demand for the Transesterification in Organic Solvents

The Pseudomonas fluorescens lipase-catalyzed transesterification of 2-methyl alkanols 1 and the 2-substituted oxiranemethanols 2 with vinyl acetate in organic solvents has been studied and the results discussed in terms of steric and electronic demand within the recently postulated models of the lipase active site.     

Production of optically active esters and alcohols from racemic alcohols by lipase-catalyzed stereoselective transesterification in non-aqueous reaction system

Microbial lipase-catalyzed transesterification between vinyl acetate and (RS)-2-octanol or (RS)-1-phenylethanol was investigated in a reaction system without addition of aqueous or organic solvents. From a screening test with various lipases, it was found that the enzymes from Pseudomonas species could efficiently catalyze the reaction, and R-enantiomers of the racemic alcohols were preferentially esterified by them. Enantiomeric purities of the optically active alcohols (S) and esters (R) obtained from (RS)-1-phenylethanol by the stereoselective transesterification of these lipases were all more than 95%.     

Lipase-catalyzed transesterification procedure for the resolution of non-protein amino acids

Summary A number of non-protein amino acids were resolved through the lipase-catalyzed enantioselective transesterification of the 2,2,2-trifluoroethyl esters of their N-benzyloxycarbonyl derivatives with methanol in organic solvents.     

Enzymatic synthesis of the acrylic esters: a comparative study

Various acrylic esters were synthesized by Candida cylindracea lipase (CCL) catalysed transesterification with different alcohols. A comparative study was carried out using 2,3-butanedione mono-oxime acrylate and vinyl acrylate as acylating agents. The rate of conversion was faster when oxime acrylate was used as acylating agent as compared to vinyl acrylate. Effect of solvents on the rate of conversion was studied and diisopropyl ether (DIPE) was proved to be a better solvent as compared to CHCl₃ and THF. The effect of various structural aspects of the substrates on the rate of conversion was studied. Among the linear alcohols studied, ease of conversion was found to be in the order of n-octanol>n-hexanol>n-butanol. Up to 80% conversion was achieved in the case of cyclohexyl methanol.     

Designing cross-linked lipase aggregates for optimum performance as biocatalysts

Cross-linked enzyme aggregates (CLEAs) are prepared by precipitation of an enzyme and then chemical cross-linking the precipitate. Three CLEAs of lipase with glutaraldehyde concentrations of 10 mM (CLEA A), 40 mM (CLEA B) and 60 mM (CLEA C) were prepared. Studies show that there is a trade-off between thermal stability vs transesterification/hydrolysis rate vs enantioselectivity. The initial rates for transesterification of β-citronellol for the uncross-linked enzyme and CLEAs A, B and C were 243, 167, 102 and 40 µmol mg^-1 h^-1, respectively. Their thermal stabilities in aqueous media, as reflected by their half-life values at 55°C, were 6, 9, 13 and 16 h, respectively. The enantioselectivity, E values (for kinetic resolution of β-citronellol by transesterification) were 19, 74, 11 and 6, respectively. These results show that CLEA C was the most thermostable; the uncross-linked enzyme was best at obtaining the highest transesterification rate; and CLEA A was best suited for the enantioselective synthesis. Scanning electron microscopy (SEM) showed that the morphology of CLEA was dependent upon the extent of cross-linking.     

Enantioselective lipase-catalyzed kinetic resolution of N-(2-ethyl-6-methylphenyl)alanine

The enzyme (BSL2), a highly active lipase expressed from newly constructed strain of Bacillus subtilis BSL2, is used in the kinetic resolution of N-(2-ethyl-6-methylphenyl)alanine from the corresponding racemic methyl ester. Reaction conditions are optimized to enhance the enantioselectivity. The effects of various racemic alkyl esters, substrate concentration, operating temperature, pH of the aqueous medium and organic solvents on activity and enantioselectivity of BSL2 for kinetic resolution are also studied. A high enantiomeric ratio (E = 60.7) is reached in diisopropyl ether/water (10%, v/v) and the enantioselectivity is about 22-fold higher than that in pure buffered aqueous solution. The results show that the reaction medium greatly influences BSL2 reaction and its enantioselectivity in the hydrolysis of racemic methyl ester.     

Kinetic modeling of immobilized-lipase catalyzed transesterification of n-octanol with vinyl acetate in non-aqueous media

Organic esters are employed as solvents, fragrance, flavors, and precursors in a variety of industries. Particularly, aliphatic esters are greatly used in flavor industry, mainly as fixatives and modifiers, and aromatic esters in fragrance compositions. Esters are produced by a variety of methods among which esterification and transesterification with acid catalysts under reflux conditions are prominent. The use of biocatalysts provides an opportunity for carrying out reactions under milder conditions leading to better quality products suitable in fragrance and flavor industry. Transesterification of n-octanol with vinyl acetate was studied at 30 °C as a model reaction by employing different lipases as catalysts such as Psedomonas species lipase immobilized on diatomite, free Candida rugosa lipase. Novozym 435 (lipase B from Candida antarctica; immobilized on macro-porous polyacrylic resin beads) and Lipozyme IM 20 (Mucor miehei lipase immobilized on anionic resin). Novozym 435 was found to be the most active catalyst in heptane as a solvent. A conversion of 82% with 100% selectivity of n-octyl acetate was obtained at 30 °C in 90 min using equimolar quantities of the reactants with 0.833 g l⁻¹ of Novozym 435. Transesterification of other alcohols such as n-decanol, benzyl alcohol, cinnamyl alcohol, 2-ethyl-1-hexanol, 1-phenyl ethyl alcohol, and 2-phenyl ethyl alcohol was also studied with vinyl acetate. The analysis of the initial rate data and progress curve data showed that the reaction obeys the ternary complex bi–bi mechanism with inhibition by n-octanol. The experimental and theoretical values matched very well.

The order of transesterification reactivity of vinyl acetate with various alcohols in presence of Novozym 435 under otherwise identical conditions at 30 °C was found to be as follows:

n-octanol>n-decanol>benzylalcohol>cinnamylalcohol>2-ethyl-1-hexanol>2-phenylethylalcohol>1-phenylethylalcohol.

     

Conductance and relaxations of gelatin films in glassy and rubbery states

The dielectric constant, ′, and the dielectric loss, ″, for gelatin films were measured in the glassy and rubbery states over a frequency range from 20 Hz to 10 MHz; ′ and ″ were transformed into M* formalism (M*=1/(′−i″)=M′+iM″; i, the imaginary unit). The peak of ″ was masked probably due to dc conduction, but the peak of M″, e.g. the conductivity relaxation, for the gelatin used was observed. By fitting the M″ data to the Havriliak–Negami type equation, the relaxation time, τ_HN, was evaluated. The value of the activation energy, E_τ, evaluated from an Arrhenius plot of 1/τ_HN, agreed well with that of E_σ evaluated from the DC conductivity σ₀ both in the glassy and rubbery states, indicating that the conductivity relaxation observed for the gelatin films was ascribed to ionic conduction. The value of the activation energy in the glassy state was larger than that in the rubbery state.     

A computational formula for heat influx in animal experiments

1. 1.The bahavioural paradigm in which cold-exposed animals can work for pulses of infrared radiation has been extensively used in the literature, but a formula to calculate the amount of heat obtained has not been advanced.

2. 2.This paper describes a computational formula for heat influx in rats: E = 3.64 · 10⁻⁶ · n · d · I · M^0.6 where E is heat influx (kJ), n is number of rewards, d is reward duration (sec), I is irradiance (mW/cm²), and M is body mass (g).

Thermodynamics of the lipase-catalyzed esterification of glycerol and n-octanoic acid in organic solvents and in the neat reaction mixture

The thermodynamics of the lipase-catalyzed esterification of glycerol with n-octanoic acid have been investigated with acetonitrile, benzene, and toluene as solvents and in the neat reaction mixture (no organic solvent added). This esterification reaction leads to five products: 1-monooctanoyl glycerol, 2-monooctanoyl glycerol, 1,2-dioctanoyl glycerol, 1,3-dioctanoyl glycerol and 1,2,3-trioctanoyl glycerol. This, in turn leads to a total of 12 reactions. Values of the equilibrium constants for these reactions have been measured (HPLC, GC, and LC/MS) at 37°C in the above mentioned media. The equilibrium constants range from 0.9 to 20.7, 0.20 to 8.0, 0.23 to 10.0, and 0.57 to 2.2 in acetonitrile, benzene, toluene, and neat media, respectively. Relative standard molar Gibbs free energies of formation Δ_fG_m⁰ of 1-monooctanoyl glycerol, 2-monooctanoyl glycerol, 1,2-dioctanoyl glycerol, 1,3-dioctanoyl glycerol and 1,2,3-trioctanoyl glycerol in the organic solvents and in the neat reaction mixture have been calculated and used to compactly summarize the thermodynamics of these reactions. The results show an approximate correlation with the permittivities of the solvents.