Preparation of an optically pure secondary alcohol of synthetic pyrethroids using microbial lipases

Summary Enzyme-catalysed hydrolysis of esters of 4-hydroxy-3-methyl-2-(2-propynyl)-cyclopent-2-enone (HMPC) was examined for the preparation of the optically pure alcohol moiety of synthetic pyrethroids. Among microorganisms and lipases tested, some bacterial lipases hydrolysed the ester of HMPC with high enantioselectivity and high reaction rate. Arthrobacter lipase gave the optically pure (R)-HMPC at 50% hydrolysis in a two-liquid phase reaction system of water and the insoluble substrate. The hydrolysis proceeded even at a substrate concentration of 80w/v%. The enantioselectivity was not changed with the chain length of the acid moiety of the esters. By combination of the enzymatic resolution with a chemical inversion of the (R)-alcohol, an efficient proess was developed for the total conversion of racemic HMPC to (S)-HMPC, which is an important alcohol for preparation of an insecticidallyactive synthetic pyrethroid.Biological preparation of an optically active alcohol. Part I     

Relative rates of hydrolysis by rat pancreatic lipase of esters of C2-C18 fatty acids with C1-C18 primary n-alcohols

The rate at which rat pancreatic lipase (glycerol-ester hydrolase, EC 3.1.1.3) hydrolyzes the esters of primary n-alcohols containing from 1 to 18 carbon atoms with fatty acids containing from 2 to 18 carbon atoms was determined. The speed of hydrolysis was influenced, apparently independently, by both the acyl and the alkyl chains. With respect to the fatty acid moiety, the esters of dodecanoic acid were usually split at the most rapid rate. Esters of butyric acid were the next most susceptible. In the case of the alcohol moiety, esters of heptyl alcohol were hydrolyzed most rapidly. On the basis of the pattern of the relative rates of hydrolysis, it is proposed that the influence of the alcohol component is a result of its orienting the ester molecule at the oil/water interface. The fatty acid effect is attributed to enzyme-substrate specificity.     

On the specificity of porcine elastase.

The specificity of porcine elastase (EC 3.4.4.7) has been studied. Ethyl esters derived from benzoyl amino acids with straight side chains are better substrates than those with branched side chains; the best substrate is norvaline ester. In the series of benzoylalanine alkyl esters the alcohol moiety markedly affects the susceptibility. The benzyl ester was found to be the best nonactivated substrate derived from monomeric amino acid. With elastase acylation is rate limiting, in contrast to chymotrypsin and trypsin where deacylation is generally the rate determining step with specific ester substrates.     

An Efficient Synthetic Method for (±)-Malyngolide and Its Optical Resolution

A convenient synthetic method for (±)-malyngolide (1), an antibiotic from the marine bluegreen alga Lyngbya majuscula GOMONT, is described. The compound (±)-1 was synthesized from 2-methylglutaric anhydride (starting material) in four steps, involving the TBHP-based osmium-catalyzed procedure for vicinal dihydroxylation of the methylene unit in unsaturated carboxylic acid as the key step. The optical resolution of (±)-l was accomplished by HPLC, in which diastereomeric (+)-2-(4-chlorophenyl)isovaleric acid [(+)-CPIA] esters of (±)-l derived from (+)-CPIA-C1 could be easily separated, and subsequent hydrolysis of each ester gave the enantiomeri-cally pure (?)-l and (+)-l, respectively. A synthetic method for α-methylene malyngolide, which is to be expected for biological activity, is also described.     

The First Synthesis of 4′-Branched 5′-Deoxycarbocyclic 9-Deazaadenosine and Phosphonic Acids as Antiviral Agents

The first synthetic route to 4′-trifluoromethylated 5′-deoxycarbocyclic-9-deazaadenosine analog and its phosphonic acid derivatives was described from α-trifluoromethyl-α,β-unsaturated ester. The C–C bond connection between cyclopentane and base moiety was accomplished using Knoevenagel type condensation from ketone derivative 11. Synthesized nucleoside and phosphonic acid analogs were tested for anti-HIV activity as well as cytotoxicity.     

Electrospray mass spectrometry of human hair wax esters

Wax esters extracted from human hair have been examined by capillary GC-MS and by nano electrospray ionization (ESI) mass spectrometry using a tandem quadrupole mass spectrometer. Initially, the wax esters were examined by capillary GC-MS using conventional means, thus revealing an incomplete chromatographic resolution of the complex array of >200 wax esters ranging from 28 to 40 carbons in length, including saturated/straight-chained, unsaturated/straight-chained, saturated/branched, and unsaturated/branched molecular species. ESI of wax esters produced ammonium adduct ions [M+NH4]+, and collisional activation of these ions formed abundant [RCO2H2]+ product ions. Wax esters containing a double bond in the fatty acyl or fatty alcohol portion of the molecule revealed identical behavior, suggesting little influence of the double bond on the ionization process or subsequent decomposition. The wax ester mixture was analyzed by ESI and tandem mass spectrometry using multiple reaction monitoring and neutral loss scanning. The neutral loss experiment [loss of NH3 and CH2=CH-(CH2)nCH3] was particularly effective at rapidly surveying the complex biological mixture, identifying>160 different wax esters that range from 24 to 42 total carbons.     

Synthesis of the α,ω-dicarboxylic acid precursor of biotin by the canonical fatty acid biosynthetic pathway

Biotin synthesis requires the C7 α,ω-dicarboxylic acid, pimelic acid. Although pimelic acid was known to be primarily synthesized by a head to tail incorporation of acetate units, the synthetic mechanism was unknown. It has recently been demonstrated that in most bacteria the biotin pimelate moiety is synthesized by a modified fatty acid synthetic pathway in which the biotin synthetic intermediates are O-methyl esters disguised to resemble the canonical intermediates of the fatty acid synthetic pathway. Upon completion of the pimelate moiety, the methyl ester is cleaved. A very restricted set of bacteria have a different pathway in which the pimelate moiety is formed by cleavage of fatty acid synthetic intermediates by BioI, a member of the cytochrome P450 family.     

Cellular Fatty Acid and Ester Formation by Brewers′ Yeast

The activity of alcohol acetyltransferase, bound to the cell membrane and responsible for the formation of acetate esters, was affected by the fatty acid composition of the cell membrane. When saturated fatty acids, which only slightly inhibit alcohol acetyltransferase activity, were in-corporated into the cell membrane, the enzyme activity and ester formation were only slightly affected. On. the other hand, when unsaturated fatty acids, which strongly inhibit the enzyme activity, accumulated in the cell membrane, ester formation was suppressed with inhibition of the enzyme activity. The mechanism of formation of acetate esters by brewers′ yeast was explained by the alcohol acetyltransferase activity under the influence of the fatty acid composition of the cell membrane.     

Enhancement of Enantioselectivity in the Bacillus subtilis Protease-catalyzed Hydrolysis of N-free Amino Acid Esters using the Ester Grouping-modification Approach

The generality of enantioselectivity enhancement through the modification of the alcohol moiety of a substrate ester was ascertained, for in the Bacillus subtilis protease-catalyzed hydrolysis of N-unprotected amino acid esters the enantioselectivity was enhanced largely by switching the conventional methyl ester to esters with a longer alkyl chain such as the isobutyl ester (from E = 3 to E = 130–170 in the case of 4-fluorophenylalanine esters) as in the enzymatic hydrolysis mediated by Aspergillus oryzae protease. There was indeed a profound dependence of E on the nature of the ester grouping.     

Structure-activity Relationships of Fungicidal N-Benzoylanthranilic Esters

The antifungal activity of 37 N-(methoxy-substituted benzoyl)anthranilic esters was tested on the powdery mildew of barley caused by Erysiphe graminis by the pot test. Among the methyl N-(methoxy-substituted benzoyl)anthranilates tested, 3,4-dimethoxybenzoyl derivative exhibited the highest activity. The variation in fungicidal activity of N-(3,4-dimethoxybenzoyl)anthranilic esters was shown to be related with variation in hydrophobicity and the electronic property of the alcohol moiety of the ester. The branching at the α-position of the alcohol moiety of the ester was detrimental to the activity.     

Esterase activity and release of ethyl esters of medium-chain fatty acids by Saccharomyces cerevisiae during anaerobic growth.

During anaerobic fermentation, Saccharomyces cerevisiae releases large amounts of medium-chain fatty acids (MCFAs) and related ethyl esters which are very important for aromatic quality of fermented beverages. The physiological function of ester synthesis is not yet understood. As MCFAs are toxic, their conversion to esters has been proposed to be a detoxification mechanism. Esterases possess ester synthesizing ability. Throughout an anaerobic fermentation of a lipid-free synthetic medium carried out with a S. cerevisiae strain selected for wine making, we have monitored MCFA and ethyl ester production and, at the same time, measured growth and esterasic activity of intact cells. Because no correlation was found between the concentration of each fatty acid and its ethyl ester, there is no evidence that ester synthesis reduces the toxicity of MCFAs. Esterasic activity did not show any correlation with ester synthesis, but it was related to the release of MCFAs. A model is proposed in which ester synthesis is a consequence of the arrest of lipid biosynthesis resulting from a lack of oxygen. Under these conditions, an excess of acyl coenzyme A is produced, and acyl esters are formed as secondary products of reactions aimed at recovering free coenzyme A.     

Novel wax esters and hydrocarbons in the cuticular surface lipids of the red harvester ant, Pogonomyrmex barbatus

The cuticular surface lipids of the red harvester ant, Pogonomyrmex barbatus, were found to contain minor amounts of novel wax esters, in addition to the major components, hydrocarbons. The wax esters ranged in carbon number from C19 to C31 and consisted of esters of both odd- and even-numbered alcohols and acids. Each wax ester with a given carbon number eluted at several different retention times indicating possible methyl branching in either the fatty acid or alcohol moiety, or in both moieties. Each eluting peak of wax esters consisted of a mixture of wax esters of the same carbon number in which the fatty acid moiety ranged from C8 to C18, and the alcohol moiety ranged from C8 to C17. Some wax esters were largely found on the head indicating they may be of a glandular origin. The hydrocarbons consisted of: n-alkanes, C23 to C33; odd-numbered n-alkenes, C27 to C35; and the major components, methyl-branched alkanes, C26 to over C49. Notable components of the methyl-branched alkanes were 2-methyltriacontane, and the novel trimethylalkanes with a single methylene between the first and second branch points, 13,15,19-trimethylhentriacontane and 13,15,21-trimethyltritriacontane.     

Absence of carotenes and presence of a tertiary methoxy group in a carotenoid from a thermophilic filamentous photosynthetic bacterium Roseiflexus castenholzii.

We identified pigments in a thermophilic filamentous photosynthetic bacterium Roseiflexus castenholzii strain HL08. We detected neither bacteriochlorophyll (BChl) c nor carotenes in this bacterium cultured under the aerobic dark and the anaerobic light conditions, which may correspond to its lack of chlorosomes. In the cells cultured under the aerobic dark conditions, the carotenoids were derivatives of keto-gamma-carotene, and the major ones were methoxy-keto-myxocoxanthin and keto-myxocoxanthin glucoside fatty acid ester. Although the tertiary methoxy group at C-1' and the double bond at C-3',4' in the psi end group of carotenoid, such as spirilloxanthin, have only been found in purple bacteria, this was the first such report in other bacterial groups. The fatty acid moiety was composed of iso fatty acids, which were rare in the cellular lipids. In the cells cultured under the anaerobic light conditions, in addition to these keto-carotenoids, we also found non-oxidized carotenoids (derivatives of gamma-carotene). Concerning the esterifying alcohol of BChl a, we found a substantial amount of geranylgeraniol, although the major component was phytol. The existence of these pigments makes this bacterium unique among the known species in CHLOROFLEXACEAE.     

播娘蒿种子脂肪油组分的GC-MS分析

播娘蒿[Descurainia sophia（L．）Webb ex Prantl]为十字花科（Cruciferae）播娘蒿属（Descuainia Webb et Berth）1～2年生草本植物,其成熟的种子俗称南葶苈子,为临床常用中药材。葶苈子味辛、苦、大寒,具泻肺降气、祛痰平喘、行水消肿之功效。用于痰涎壅肺、喘咳痰多、胸肋胀满、胸腹水肿、小便不利、肺原性心脏病水肿等症。     

Anaerobic degradation of ethylbenzene by a new type of marine sulfate-reducing bacterium

Anaerobic degradation of the aromatic hydrocarbon ethylbenzene was studied with sulfate as the electron acceptor. Enrichment cultures prepared with marine sediment samples from different locations showed ethylbenzene-dependent reduction of sulfate to sulfide and always contained a characteristic cell type that formed gas vesicles towards the end of growth. A pure culture of this cell type, strain EbS7, was isolated from sediment from Guaymas Basin (Gulf of California). Complete mineralization of ethylbenzene coupled to sulfate reduction was demonstrated in growth experiments with strain EbS7. Sequence analysis of the 16S rRNA gene revealed a close relationship between strain EbS7 and the previously described marine sulfate-reducing strains NaphS2 and mXyS1 (similarity values, 97.6 and 96.2%, respectively), which grow anaerobically with naphthalene and m-xylene, respectively. However, strain EbS7 did not oxidize naphthalene, m-xylene, or toluene. Other compounds utilized by strain EbS7 were phenylacetate, 3-phenylpropionate, formate, n-hexanoate, lactate, and pyruvate. 1-Phenylethanol and acetophenone, the characteristic intermediates in anaerobic ethylbenzene degradation by denitrifying bacteria, neither served as growth substrates nor were detectable as metabolites by gas chromatography-mass spectrometry in ethylbenzene-grown cultures of strain EbS7. Rather, (1-phenylethyl)succinate and 4-phenylpentanoate were detected as specific metabolites in such cultures. Formation of these intermediates can be explained by a reaction sequence involving addition of the benzyl carbon atom of ethylbenzene to fumarate, carbon skeleton rearrangement of the succinate moiety (as a thioester), and loss of one carboxyl group. Such reactions are analogous to those suggested for anaerobic n-alkane degradation and thus differ from the initial reactions in anaerobic ethylbenzene degradation by denitrifying bacteria which employ dehydrogenations.     

Substrate specificity of an α-amino acid ester hydrolase produced by Acetobacter turbidans A.T.C.C. 9325

A partially purified preparation of an alpha-amino acid ester hydrolase was obtained from Acetobacter turbidans A.T.C.C. 9325, which catalyses synthesis of 7-(d-alpha-amino-alpha-phenylacetamido)-3-cephem-3-methyl-4- carboxylic acid (cephalexin) from methyl d-alpha-aminophenylacetate and 7-amino-3-deacetoxycephalosporanic acid. The enzyme preparation catalysed both cephalosprin synthesis from 7-amino-3-deacetoxycephalosporanic acid and suitable amino acid esters (e.g. methyl d-alpha-aminophenylacetate, l-cysteine methyl ester, glycine ethyl ester, d-alanine methyl ester, methyl dl-alpha-aminoiso-butyrate, l-serine methyl ester, d-leucine methyl ester, l-methionine methyl ester) and the hydrolysis of such esters. The substrate specificity of the enzyme preparation for the hydrolysis closely paralleled the acyl-donor specificity for cephalosporin synthesis, even to the reaction rates. Only alpha-amino acid derivatives could act as acyl donors. The hydrogen atom on the alpha-carbon atom was not always required by acyl donors. The hydrolysis rate was markedly diminished by adding 7-amino-3-deacetoxycephalosporanic acid to reaction mixtures, but no effect on the total reaction rate (the hydrolysis rate plus synthesis rate) was observed with various concentrations of 7-amino-3-deacetoxycephalosporanic acid. Both the hydrolytic and the synthetic activities of the enzyme preparation were inhibited by high concentrations of some acyl donors (e.g. methyl d-alpha-aminophenylacetate, ethyl d-alpha-aminophenylacetate). The enzyme preparation hydrolysed alpha-amino acid esters much more easily than alpha-amino acid derivatives with an acid-amide bond.     

The surface wax composition of the exuviae and adults of Aleyrodes singularis

Long-chain aldehydes, alcohols, hydrocarbons and wax esters were major components of the external lipids of adult Aleyrodes singularis. In exuviae, acetate esters replaced the hydrocarbons as a major component. The major long-chain alcohol and aldehyde from adults were C32 and were essentially the exclusive components of the wax particles. The major alcohol from exuviae was C26 and the aldehydes were C26, C28, C30 and C32. The major acetate esters were C28 and C30 in both adults and exuviae. There were wax esters of similar carbon number in adults and exuviae although the exuviae had a greater amount of wax esters with unsaturated fatty acids. The fatty acid and alcohol composition of the wax esters differed markedly between adults and exuviae. Wax esters of adults had similar amounts of C16, C18, C20, C22 and C24 fatty acids while those from exuviae contained largely C16 and C18. The major alcohol in the wax esters of adults was C22 and those of exuviae were C26 and C28. The distribution of fatty acids and alcohols among wax esters of varying chain length also differed between adults and exuviae: in adults C22 was the major fatty acid found in the dominant wax ester, C44 and the C22 alcohol was the major alcohol and found in wax esters C42 and C44. In exuviae C16 and C18 were the major fatty acids found in most wax esters and a C28 alcohol was the major alcohol found in wax esters C44 and C46, the two dominant wax esters in exuviae. It was clear that the difference in chemistry of the wax esters between the adults and exuviae is not evident unless the acid and alcohol moieties are characterized.     

On the relationship between methane production and oxidation by anaerobic methanotrophic communities from cold seeps of the Gulf of Mexico

The anaerobic oxidation of methane (AOM) in the marine subsurface is a significant sink for methane in the environment, yet our understanding of its regulation and dynamics is still incomplete. Relatively few groups of microorganisms consume methane in subsurface environments – namely the anaerobic methanotrophic archaea (ANME clades 1, 2 and 3), which are phylogenetically related to methanogenic archaea. Anaerobic oxidation of methane presumably proceeds via a 'reversed' methanogenic pathway. The ANME are generally associated with sulfate-reducing bacteria (SRB) and sulfate is the only documented final electron acceptor for AOM in marine sediments. Our comparative study explored the coupling of AOM with sulfate reduction (SR) and methane generation (MOG) in microbial communities from Gulf of Mexico cold seep sediments that were naturally enriched with methane and other hydrocarbons. These sediments harbour a variety of ANME clades and SRB. Following enrichment under an atmosphere of methane, AOM fuelled 50–100% of SR, even in sediment slurries containing petroleum-associated hydrocarbons and organic matter. In the presence of methane and sulfate, the investigated microbial communities produce methane at a small fraction (∼10%) of the AOM rate. Anaerobic oxidation of methane, MOG and SR rates decreased significantly with decreasing concentration of methane, and in the presence of the SR inhibitor molybdate, but reacted differently to the MOG inhibitor 2-bromoethanesulfonate (BES). The addition of acetate, a possible breakdown product of petroleum in situ and a potential intermediate in AOM/SR syntrophy, did not suppress AOM activity; rather acetate stimulated microbial activity in oily sediment slurries.     

Pyrrolic and N-oxide metabolites formed from pyrrolizidine alkaloids by hepatic microsomes in vitro: Relevance to in vivo hepatotoxicity

An analytical method of improved sensitivity has enabled measurements to be made of N-oxide as well as pyrrolic metabolites formed from a range of unsaturated pyrrolizidine alkaloids in hepatic microsome preparations. Using microsomes from livers of phenobarbitone-pretreated male Fischer rats, all 13 alkaloids tested were metabolised to both N-oxides and pyrroles. The most lipophilic alkaloids gave enhanced rates of metabolism. No consistent relationship existed between rates of N-oxide and of pyrrole formation. The two pathways appeared to be independent. The ratio of N-oxide to pyrrolic metabolites varied, depending on the type of ester: it was highest for ‘open’ diester alkaloids, lowest for 12 membered macrocyclic diesters and for monoesters. Steric hindrance by the acid moiety could account for these differences, by affecting the balance between microsomal oxidation of the amino alcohol moiety at the nitrogen and C8 positions respectively and could explain the high pyrrole yields given by some macrocyclic diesters. The levels of pyrrolic metabolites bound to liver tissues and responsible for hepatotoxicity in rats given pyrrolizidine alkaloids, did not necessarily reflect the rates of formation of such metabolites measured in vitro. In the animal additional factors could influence the formation and tissue binding of pyrrolic metabolites, including the detoxication of alkaloids by hydrolysis and the chemical reactivity and stability of the toxic metabolites. A comparison of heliotridine esters with retronecine esters showed that the 7-hydroxyl or -ester configuration had a relatively small influence on the balance between formation of pyrrolic metabolites and detoxication by N-oxidation. The results did not support any hypothesis that heliotridine esters should generally be more hepatotoxic than analogous retronecine esters. The structure of the acid moiety was likely to have at least as much influence on toxicity as the base configuration.