Resolution of racemic carboxylic acids via the lipase‐catalyzed irreversible transesterification of vinyl esters

The lipase‐catalyzed irreversible transesterification procedure using vinyl esters was applied to the resolution of racemic 2‐phenoxypropanoic acids. Aspergillus niger lipase showed high enantioselectivities and reasonable reaction rates. The enantioselectivity was found to be affected profoundly by several variables, e.g., the alcohol as nucleophile, the organic solvent used, and the reaction temperature. A gram‐scale resolution of (RS)‐2‐phenoxypropanoic acid was achieved after optimization of the reaction conditions. Then this irreversible transesterification procedure was applied to the resolution of some related 2‐substituted carboxylic acids. Thus, racemic 2‐methoxy‐2‐phenylacetic acid was resolved via the A. niger lipase‐catalyzed transesterification of the corresponding vinyl ester. 2‐Phenylpropanoic acid and 2‐phenylbutanoic acid were resolved using Pseudomonas sp. lipase. A gram‐scale resolution of 2‐phenylbutanoic acid was achieved by this procedure coupled with the porcine liver esterase‐catalyzed hydrolysis of the resulting methyl ester. Chirality 11:554–560, 1999. © 1999 Wiley‐Liss, Inc.     

Novel bacterial isolates for the resolution of esters of tertiary alcohols

Summary Resolution of a tertiary allylic terpene alcohol (linalool) employed in flavor and fragrance applications was achieved via biocatalytic hydrolysis of its acetate ester using the carboxyl ester hydrolase activity of lyophilized bacterial cells. Several strains of the genera Rhodococcus, Nocardia, Arthrobacter and Mycobacterium were found to be active. In general, the enantioselectivity was low; only with Rhodococcus ruber SM 1792 (S)-linalool was obtained in 58% e.e. (E-value ~5).     

Axially chiral N,N′‐dioxides ethers for catalysis in enantioselective allylation of aldehydes

A series of axially chiral ethers synthesized from biscarboline N,N′‐dioxides, (S)‐ 1a to (S)‐ 1n , was investigated in enantioselectivity addition reactions of allyltrichlorosilane with a series of substituted aldehydes, including bulky substituted aldehydes. High enantioselectivities (up to 96%ee) were achieved using the catalyst (S)‐ 1k at 1 mol % loading.     

Stabilization of Candida lipase against acetaldehyde by adsorption onto celite

Summary Immobilization of Candida rugosa lipase (Amano AY-30) by adsorption onto Celite 545 led to a markedly improved performance of the enzyme: Firstly, the enzyme was almost completely resistant to deactivation by acetaldehyde, which is liberated as unavoidable by-product in acyl-transfer reactions with vinyl esters, and secondly, the enantioselectivity was enhanced up to three-fold.     

Asymmetric imine-ene reactions: Diastereofacial selective reactions with chiral glyoxylate-derivedα-imino esters and asymmetric catalysis of enantiofacial selective reactions with prochiralα-imino esters

Summary The diastereofacial selective imine-ene reactions with-imino esters, prepared from (–)-8-phenylmenthyl glyoxylate, are shown to provide an efficient entry to the asymmetric synthesis of-amino acids. The feasibility study of the asymmetric catalysis is also reported on the enantiofacial selective ene reactions with prochiral-imino esters.     

Treatment of phenolic wastes byAureobasidium pullulans adhered to the fibrous supports

Summary Biological treatment of waste water containing a large amount of phenol was carried out by using a phenolassimilating fungus,Aureobasidium pullulans No. 14 adhered (semi-immobilized) to fibrous asbestos. The column reactor employed for oxidative degradation of phenol consisted of a cylindrical glass column containing plastic nets.During 27 days operation, it was observed that: 1) The phenol removal capacity of the reactor gradually increased during the first 10 days, reaching a stable level. 2) The best phenol removal capacity (50 mg phenol removed/h/ liter of reactor volume) was obtained when an artificial waste water containing up to 1,200 g/ml phenol was applied to the reactor. 3) Much higher concentrations of phenol (e.g. 1,700 g/ml) brought about a marked decrease in the phenol removal capacity (40–50 mg/h/liter). 4) Satisfactorily stable operation was achieved using the semiimmobilized mycelia ofAureobasidium pullulans, whose active state could be checked by observing the thick, black-colored biomass which is characteristic of the genusAureobasidium and covered the plastic nets inside the glass column reactor.     

Phorbol ester stimulation of phosphatidylcholine synthesis in four cultured neural cell lines: Correlations with expression of protein kinase C isoforms

Phosphatidylcholine (PtdCho) can provide lipid second messengers involved in signal transduction pathways. As a measure of phospholipid turnover in response to extracellular stimulation, we investigated differential enhancement of [³H]choline incorporation into PtdCho by phorbol esters. In C6 rat glioma and SK-N-SH human neuroblastoma cells, [³H]PtdCho synthesis was 2–4 fold stimulated by -12-O-tetradecanoylphorbol-13-acetate (-TPA) when [³H]choline was incubated simultaneously with, or 15 min prior to, -TPA treatment. By contrast, in N1E-115 mouse and SK-N-MC human neuroblastoma cells, phorbol esters had no appreciable effect on [³H]choline incorporation; however, in all cells, 200 M oleic acid enhanced PtdCho synthesis, indicating a stimulable process. Alterations by thymeleatoxin (TMT), an activator of conventional PKC isoforms (, and ), were similar to -TPA. We investigated whether expression of specific PKC isoforms might correlate with these effects of phorbol esters on PtdCho synthesis. All cell lines bound phorbol esters, had PKC activity that was translocated by phorbol esters and differentially expressed isoforms of PKC. Northern and western blot analyses, using specific cDNA and antibodies for PKC-,-,-,-,-, and-, revealed that expression of -isoform predominated in C6 and SK-N-SH cells. In contrast, TPA-responsive -isoform predominated in SK-N-MC cells. -PKC was not detected in any cells and only in C6 cells was PKC- present and translocated by -TPA treatment. PKC- was not detected in SK-N-MC cell lines but translocated with TPA treatment in the other three cell lines. PKC- was present in all cells but was unaltered by TPA treatment. Accordingly, stimulation of PtdCho turnover by phorbol esters correlated only with expression of PKC-; presence of PKC- alone was insufficient for a TPA response.Abbreviations DAG diacylglycerol - DMEM Dulbecco's modified Eagle's medium - dPPA 12-deoxyphorbol-13-phenylacetate-20-acetate - PKC protein kinase C - cPKC conventional PKC - PtdCho phosphatidylcholine - TPA 12-O-tetradecanoylphorbol-13-acetate - TMT thymeleatoxin     

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.     

Spontaneous excitatory postsynaptic currents in crayfish neuromuscular junctions in the absence and presence of serotonin and 3,4-diaminopyridine

Summary Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded under voltage clamp in short fibres (l0.6mm) from opener muscles and the contractor epimeralis muscle of small crayfish. From the amplitude distributions of sEPSCs which could be approximated by a Gaussian function, a mean amplitudeã= –1.16 nA±0.28 (SE) was found for sEPSCs in 16 fibres of the claw opener voltage clamped toE=–60 mV (19–22 °C). In the opener of the first walking leg and in the contractor epimeralis muscleã=s-1.1 nA±0.21 (SE;n= 6, –100 mVE–60 mV, 5–10 °C) andã= –2.0 nA±0.2 (SE;n=4, E=–60 mV, 19–22 °C) were obtained. On average about 300–500 synaptic channels were estimated to open during a sEPSC. Giant sEPSCs (gsEPSCs) were also observed. The amplitudes of gsEPSCs were up to 14 times larger than the amplitude of an average normal sEPSC. Moreover, the lifetime of gsEPSCs was up to about 3 times longer than that of sEPSCs. Like sEPSCs, gsEPSCs could not be abolished by 0.1 mol/l tetrodotoxin. The rate at which sEPSCs and gsEPSCs occurred could be markedly enhanced by serotonin (1 mol/l) and 3,4-diaminopyridine (1 mol/l)Abbreviations sEPSCs spontaneous excitatory postsynaptic currents - gsEPSCs giant spontaneous excitatory postsynaptic currents - sIPSCs spontaneous inhibitory postsynaptic currents - gsIPSCs giant spontaneous inhibitory postsynaptic currents - 5-HT 5-hydroxytryptamine - 3,4-DAP 3,4-diaminopyridine - time constant of exponential decay of sEPSCs or gsEPSCs - t _B50 lifetime of sEPSCs or gsEPSCs given by the width of these currents at 50% of their amplitude; ã amplitude of sEPSCs or gsEPSCs - i current amplitude evoked by opening of single glutamate-activated channels - z number of channels open at the peak of an average sEPSC This investigation was supported by the Deutsche Forschungsgemeinschaft. Project Fi 305/1-3     

Highly efficient transformation and regeneration of aspen plants through shoot-bud formation in root culture

The natural capacity of aspen (Populus tremula L.) roots for direct shoot-bud regeneration was harnessed to establish a highly efficient transformation and regeneration procedure that does not require a pre-selection stage on antibiotics. Aspen stem segments were transformed using wildtype Agrobacterium rhizogenes (LBA9402) with the binary p35SGUSINT plasmid carrying the genes coding for -glucuronidase (GUS) and neomycin phosphotransferase II. High levels of transient GUS expression were found in the basal cut surface of 87% of the segments, and 98% of these formed well-developed adventitious roots. Proliferating root cultures were established in liquid culture, and GUS expression was found in 75% of the roots. Shoot-bud regeneration in root cultures was very high: 99% of the roots yielded shoot-buds (4.3 buds per root), of which 91% expressed GUS. Southern blot analysis and polymerase chain reaction confirmed the transgenic nature of the plants expressing GUS. Kanamycin resistance of transformants was tested with respect to callus growth and bud regeneration. Callus from transgenic plants exhibited a high growth rate in the presence of up to 100 g/l kanamycin, and bud regeneration from transformed roots occurred in the presence of up to 30 g/l kanamycin. Callus and buds from control (non-transformed) plants failed to proliferate or regenerate, respectively, in the presence of kanamycin at concentrations above 10 g/l. Ninety-four independent clones from different transformation events were established, of which 52 were phenotypically true-to-type.Abbreviations NAA -naphthaleneacetic acid - BA 6-benzylaminopurine - GUS -glucuronidase - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction - EtOH ethanol - CTAB cetyltrimethylammonium bromide - SDS sodium dodecyl sulfate - NOS nopaline synthase - CaMV cauliflower mosaic virus     

Purification and Characterization of an Esterase Involved in Poly(vinyl alcohol) Degradation by Pseudomonas vesicularis PD

An esterase catalyzing the hydrolysis of acetyl ester moieties in poly(vinyl alcohol) was purified 400-fold to electrophoretic homogeneity from the cytoplasmic fraction of Pseudomonas vesicularis PD, which was capable of assimilating poly(vinyl alcohol) as the sole carbon and energy source. The purified enzyme was a homodimeric protein with a molecular mass of 80 kDa and the isoelectric point was 6.8. The pH and temperature optima of the enzyme were 8.0 and 45°C. The enzyme catalyzed the hydrolysis of side chains of poly(vinyl alcohol), short-chain p-nitrophenyl esters, 2-naphthyl acetate, and phenyl acetate, and was slightly active toward aliphatic esters. The enzyme was also active toward the enzymatic degradation products, acetoxy hydroxy fatty acids, of poly(vinyl alcohol). The K _m and V _max of poly(vinyl alcohol) (degree of polymerization, 500; saponification degree, 86.5-89.0 mol%) and p-nitrophenyl acetate were 0.381% (10.6 mM as acetyl content in the polymer) and 2.56 μM, and 6.52 and 12.6 μmol/min/mg, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate at a concentration of 5 mM, which indicated that the enzyme was a serine esterase. The pathway for the metabolism of poly(vinyl alcohol) is also discussed.     

Enzymatic resolution of 2-phenoxy-1-propanols through the enantioselective acylation mediated by Achromobacter sp. lipase

2-(Substituted phenoxy)-1-propanols, e.g. 2-(4-chlorophenoxy)-1-propanol, belonging to primary alcohols with an oxygen atom at the stereocenter, were resolved with moderate to good enantioselectivity, as judged by the value of enantiomeric ratio E (up to 27), through the enantioselective acylation with vinyl butanoate mediated by the little-known lipase from Achromobacter sp. in diisopropyl ether, after the examination of potential factors affecting the reaction such as organic solvents and acyl donors.     

Synthesis of flavor and fragrance esters using <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase

Candida antarctica lipase fraction B (CAL-B) showed substrate specificity in the synthesis of esters in hexane involving reactions of short-chain acids having linear (acetic and butyric acids) and branched chain (isovaleric acid) structures, an unsaturated (tiglic acid) fatty acid, and phenylacetic acid with n-butanol and geraniol. The variation in the conversion to the esters was ca. 10%. Similar results were observed in a study of the alcohol specificity of the enzyme for esterification of acetic and butyric acids with four alcohols: n-butyl, isopentyl, 2-phenylethyl, and geraniol. Enantioselectivity of CAL-B in hexane with a range of chiral -substituted or -substituted carboxylic acids and n-butyl alcohol was analyzed. The results show that CAL-B can be employed as a robust biocatalyst in esterification reactions due to the high conversions obtained in the synthesis of short-chain flavor esters in an organic solvent, although this enzyme exhibited modest enantioselectivity with chiral short-chain carboxylic acids.     

New Photosensitizers of Bacteriochlorin Series for Photodynamic Cancer Therapy

New derivatives of bacteriochlorophyll a bearing an extra glutarimide exocycle were synthesized, and their reactivity was studied. Acetyl group in 3-acetyl-2,7,12,18-tetramethyl-8-ethyl-13,15-dicarboxy-17-carboxyethyl-7,8,17,18-tetrahydroporphyrin (bacteriochlorin p) was chemically modified into -hydroxyethyl and vinyl groups. A simple method of preparation of vinylbacteriopurpurin esters under the catalysis by p-toluenesulfonic acid was proposed. The resulting compounds exhibit a high adsorption in the visible and near IR areas of electronic spectra, a reasonable stability, and amphiphilic properties and, therefore, may be regarded as promising photosensitizers for the photodynamic cancer therapy.     

The food and parasites of fish in some deep basins of northern L. Päijänne

The composition of the fish stock, food and macroparasites were studied in eleven basins (22–100 m) of Lake Jyväsjärvi and North Päijänne in August–September 1976. The fishing was done by means of a series of nets (meshes 15, 21 and 35 mm) laid on the bottom overnight. No fish were found in the two northernmost basins owing to bad oxygen conditions caused by waste waters. Smelt and burbot were the most abundant fish in the catches in other basins but the vendace was rare. Relict crustaceans and some deep water copepods (e.g. Heterocope borealis) played an important role in the food of fish in all basins.Parasites spreading via relict crustaceans were abundant in smelt and burbot, but absent in vendace and ruffe. The most common parasite in smelt was Cystidicola farionis, in burbot Echinorhynchus borealis, in ruffe Triaenophorus nodulosus and in vendace the gill parasite Ergasilus sieboldi. Females of a relatively rare copepod Salmincola lotae were found in the oral cavity of five burbot (at depths of 50 and 100 m).     

Purification and characterization of hydroxycinnamoyl-coenzyme A: ω-hydroxypalmitic acid O-hydroxycinnamoyltransferase from tobacco (Nicotiana tabacum L.) cell-suspension cultures

An acyltransferase hydroxycinnamoyl-Coenzyme A: -hydroxypalmitic acid O-hydroxycinnamoyltransferase (HHT; EC 2.3.1.-), which transfers hydroxycinnamic acids from hydroxycinnamoyl-CoA thioesters to several hydroxylated fatty acid derivatives, was characterized from tobacco (Nicotiana tabacum L. cv. Xanthi nc) cell-suspension cultures. It exhibited the same properties as the enzyme previously detected in wound-healing potato tuber discs (Lotfy et al., 1994, Phytochemistry 35: 1419–1424), and especially a marked specificity for -hydroxypalmitic acid and feruloyl-CoA. It was purified 300-fold to near homogeneity from late logarithmic-phase cell suspensions. The apparent molecular mass of the native protein was 55 kDa and its isoelectric point, estimated by electrofocusing, was 4.6. The purified enzyme conjugated ferulic acid to -hydroxypalmitic acid and to 1-tetradecanol, its main lipidic substrates, suggesting that the same enzyme probably synthesizes the different esters of 1-alkanols and of -hydroxy fatty acids which are formed in vitro.Abbreviations ABA abscisic acid - IEF isoelectric focusing - HHT hydroxycinnamoyl-Coenzyme A: -hydroxypalmitic acid O-hydroxycinnamoyltransferase - pI isoelectric point     

Conformation of the transmembrane domain of the epidermal growth factor receptor

The transmembrane domain of growth factor receptors, such as the epidermal growth factor receptor (EGFR) and the relatedc-erbB-2/neu oncogene protein, has been implicated in the process of receptor dimerization and mitogenic signal transduction, and hence in cellular transformation and oncogenesis. Amino acid substitutions in the transmembrane domain of thec-erbB-2/neu protein that cause a transforming effect may exert this effect through a conformational change from a bend conformation to an-helical structure in this region of the protein, but similar amino acid substitutions at homologous positions in the transmembrane domain of the EGFR (e.g., ValGlu at position 627) fail to have a transforming effect. To examine whether this failure may be due to structural effects, we have used conformational energy analysis to determine the preferred three-dimensional structures for the nonapeptide sequence of the transmembrane domain of the EGFR from residues 623–631 with Val or Glu at position 627. The global minimum energy conformations of both nonapeptides were found to be non--helical with bends at positions 624–625 and 627–628. The failure of the ValGlu substitution to produce a conformational change to an-helix in this region may be responsible for its lack of transforming effect. However, the presence of higher energy-helical conformations for the nonapeptide from the normal EGFR may provide an explanation for the presence of a transforming effect from overexpression of the EGFR.     

Competition between the sibling species Tisbe holothuriae and T. battagliai (Harpacticoida)

The influence of salinity on the performance of the sibling species Tisbe holothuriae and T. battagliai in pure and mixed cultures was studied, using laboratory stocks adapted to 32 for over 175 generations. Cohort studies show that T. holothuriae has higher growth rates (R_o and r) at 32, T. battagliai at 20 The latter's life cycle is much less affected by the difference in salinity. De Wit replacement series were used to study competitive interactions. Without water renewal, T. holothuriae eliminates its sibling species in less than 2 generations, apparently through chemical interference. With water renewal, i.e. when exploitation competition becomes relatively more important, T. holothuriae still proves superior at 27 but the two species are competitively almost equal at 20. The two species cooccur in situ during autumn, but their differential predominance at different sites is not explained by the effect of salinity.     

Lipase-catalyzed synthesis of vitamin C fatty acid esters

Fatty acid esters of vitamin C (ascorbic acid) where synthesized in a mainly solid-phase system in the presence of small amounts of organic solvent (acetone or t-butanol) catalyzed by immobilized lipase B from Candida antarctica.Highest reaction rates and yields of isolated products were obtained using fatty acid vinyl esters, e.g., 6-O-palmitoyl-l-ascorbic acid was obtained in 91% isolated yield after 48 h. As vitamin C and its esters are very sensitive to oxidation, a mild extraction method for the isolation of reaction products was developed.     

Uptake of sewage derived nitrogen by<Emphasis Type="Italic"> Ulva rigida</Emphasis> (Chlorophyceae) in Bahía Nueva (Golfo Nuevo,Patagonia, Argentine)

Uptake kinetics of nitrogen derived from sewage–seawater mixtures (2.5–20% v/v effluent) were determined in the laboratory for Ulva rigida (Chlorophyceae) native from Bahía Nueva (Golfo Nuevo, Patagonia, Argentine). In terms of nitrogen concentration, experimental enrichment levels varied between 53.7 and 362.3M of ammonium and between 0.77 and 6.21M of nitrate+nitrite. Uptake rates were fitted to the Michaelis–Menten equation, with the following kinetic parameters: ammonium: V_max = 591.2molg^–1h^–1, K _s=262.3M, nitrate+nitrite: V _max=12.9molg^–1h^–1, K _s=3.5M). Both nutrients were taken up simultaneously, but ammonium incorporation was faster in all cases. The results show a high capability of Ulva rigida to remove sewage-derived nitrogen from culture media. In the field, most of the nitrogen provided by the effluent would be tied up in algal biomass, supporting low nitrogen levels found at a short distance away from the source.