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.     

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     

A cold active (2R,3R)-(−)-di-O-benzoyl-tartrate hydrolyzing esterase from Rhodotorula mucilaginosa

In a screening procedure a pink-colored yeast was isolated from enrichment cultures with (2R,3R)-(−)-di-O-benzoyl-tartrate (benzoyl-tartrate) as the sole carbon source. The organism saar1 was identified by morphological, physiological, and 18S ribosomal DNA/internal transcribed spacer analysis as Rhodotorula mucilaginosa, a basidiomycetous yeast. During growth the yeast hydrolyzed the dibenzoyl ester stoichiometrically to the monoester using the separated benzoate as the growth substrate, before the monoester was further cleaved into benzoate and tartrate, which were both metabolized. The corresponding benzoyl esterase was purified from the culture supernatant and characterized as a monomeric glycosylated 86-kDa protein with an optimum pH of 7.5 and an optimum temperature of 45 °C. At 0 °C the esterase still exhibited 20% of the corresponding activity at 30 °C, which correlates it to psychrophilic enzymes. The esterase could hydrolyze short chain p-nitrophenyl-alkyl esters and several benzoyl esters like benzoyl-methyl ester, ethylene-glycol-dibenzoyl ester, phenyl-benzoyl ester, cocaine, and 1,5-anhydro-d-fructose-tribenzoyl ester. However feruloyl-ethyl ester was not hydrolyzed. The activity characteristics let the enzyme appear as a promising tool for synthesis of benzoylated compounds for pharmaceutical, cosmetic, or fine chemical applications, even at low temperatures.     

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.     

Fungicidal Activity of N-Benzoylanthranilates and Related Compounds

Methyl N-(substituted benzoyl)anthranilates were found to possess inhibitory activity against the powdery mildew of cucumber caused by Sphaerotheca fuliginea. Both the anthranilate and the N-benzoyl moieties were essential for this type of fungicidal activity. Substitution at the 2- and 4-positions of the N-benzoyl group was unfavorable to the activity except for the 4-methoxy group. Substitution at the 3-position varied the fungicidal activity to various extents. The variation in the activity of 3-substituted derivatives was analyzed quantitatively with substituent parameters and regression analysis indicating that the variation in the steric dimension of substituents was most responsible for the activity.     

Attraction of the German cockroach to cyclohexyl alkanoates and n-alkyl cyclohexaneacetates

Attraction of the German cockroach, Blattella germanica, to esters was tested in a heavily infested room. Sticky traps of cardboard baited with hexyl hexanoate and pentanoate captured higher numbers than an empty control. Recluction or extension of the alcohol moiety even by a C₁ unit resulted in a decrease of activity. The suggested importance of the C₆ moiety which is known to make a ring form with the least effort led to examination of the activity of the corresponding cyclohexanol esters; the result was some tenfold increase in activity. A further tenfold increase in attractiveness was attained by inverting the O and C adjacent to the ester carbonyl function, i.e. by modifying cyclohexyl pentanoate into propyl cyclohexaneacetate.     

Plant Growth Inhibition and Membrane Penetration by a Homologous Series of Dichlorobenzoate Esters

The homologous series of n-alkyl esters (C₁–C₁₀) of 3,4-dichlorobenzoic acid was synthesized and their effects in inhibiting the growth of Nicotiana meristems were studied. The inhibition of growth was considered in terms of penetration of chemicals into the plant tissue and subsequent cell membrane disruption. Penetration was investigated by applying the emulsified ester to the meristem and then measuring the compound recovered with the isolated surface lipids. Decreasing amounts of 3,4-dichlorobenzoate esters penetrated into the plant as the alkyl chain length of the ester moeity was increased. Essentially no penetration occurred with the n-C₇ through C₁₀ esters tested. The effect of the ester homologues on cell membranes was studied by measuring the efflux of betacyanin from beet root cells. Decreasing amounts of pigments were released as the alkyl chain length of the ester was increased. Minimal cell membrane disruption was found for the C₇–C₁₀ esters. Inhibition of the plant meristem may result from the more rapid penetration of the short chain ester homologues into the plant.     

Synthesis of N-hydroxycinnamoyl amide derivatives and evaluation of their anti-oxidative and anti-tyrosinase activities

Twelve N-hydroxycinnamoyl amino acid amide ethyl esters (CAES) were synthesized by using l-amino acid ethyl ester hydrochloride and corresponding cinnamic acid (ferulic acid, acetylferulic acid and caffeic acid) as raw materials in the presence of a catalytic amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide-hydrochloride (EDC) and 1-hydroxybenzotriene (HOBt). The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activities of CAES were evaluated. The anti-tyrosinase activities of N-feruloyl amino acid ethyl esters and the hydroxyl (OH) free radical scavenging activities of N-caffeoyl amino acid ethyl esters were also examined. DPPH free radical scavenging activity was shown in all CAES, of which N-caffeoyl amino acid ethyl esters demonstrated higher radical scavenging activity than N-feruloyl amide derivatives, and (E) -N-(caffeic acid)-l-glycinate ethyl ester (c₅) had the strongest ability to scavenge free radicals with an IC₅₀ value of 18.6 µM. The acetylferuloyl amino acid esters exhibited the highest tyrosinase inhibition activity among the tested amides.     

Short chain flavour esters synthesis by microbial lipases

Summary The peparative synthesis of 35 short chain flavour esters by lipases fromMucor miehi, Aspergillus sp.,Candida rugosa andRhizopus arrhizus was investigated in organic media. Acetic, propionic, butyric, valeric and caproic acids, as well as methanol, ethanol, butanol, i-pentanol, hexanol, citronellol and geraniol were used as substrates. Most of the esters were synthesized in good yield by at least one of the lipase preparations tested. Different conversion yields were observed according to the lipase specificity toward the acid or the alcohol moiety of the ester. Methyl- and ethyl acetates were also produced by changing the organic solvent. Enzymatic catalysis in organic solvent is thought to be a valuable method for preparative synthesis of flavour esters.     

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.     

Activation of sulfonate ester based matrix metalloproteinase proinhibitors by hydrogen peroxide

This study details the development of matrix metalloproteinase inhibitor prodrugs (proMMPi) that are activated in the presence of reactive-oxygen species (ROS). Conventional matrix metalloproteinase inhibitors (MMPi) utilize a zinc-binding group (ZBG) that chelates to the catalytic zinc(II) ion of matrix metalloproteinases (MMPs) to inhibit their activity. To create ROS-sensitive prodrugs, sulfonate esters were used as a protecting group for the ZBG to block their metal binding ability. Surprisingly, these sulfonate esters were found to be cleaved by H₂O₂ only when the ZBG contained an N-oxide donor atom moiety. Sulfonate ester derivatives of full-length MMPi based on these ROS-triggerable systems were synthesized. It was found that proMMPi with sulfonate ester protecting groups showed relatively high rates of cleavage in the presence of H₂O₂ to release the active MMPi. In vitro MMP inhibition studies confirmed a significant increase in inhibitory activity of proMMPi upon addition of H₂O₂, demonstrating the use of sulfonate esters to act as cleavable triggers for ROS-activated prodrugs.     

The Synthesis of Some 5-Vinyluracil-Nucleoside Analogues

Abstract

The reaction of 5-iodouridine with esters of acrylic acid in the palladium-catalysed Heck reaction was used to generate a series of esters of the acid (E) -5- (2-carboxyvinyl)uridine in poor to moderate yield. An alternative method starts from the acid by protection of the sugar moiety followed by in situ generation of the acid chloride then ester formation followed by simultaneous sugar deprotection. Treatment of the methyl ester with ammonia and methylamine gave the corresponding amides, while treatment with dimethylamine gave 5-(1-dimethylamino-2-carboxy)ethyluridine as the major product.     

Lipase-catalyzed enantioselective hydrolysis of N-protected racemic non-protein amino acid esters

Porcine pancreatic lipase (PPL)-catalyzed enantioselective hydrolysis of N-benzyloxycarbonyl-dl-amino acid esters (Z-dl-AA-ORs) was studied for the optical resolution of a variety of non-protein amino acids. The ester moiety (R) of the substrate affected the rate of hydrolysis significantly. The glyceryl (Gl) and carbamoylmethyl (Cam) esters were found to be highly reactive substrates. The hydrolysis of the Gl esters (Z-dl-AA-OGls) of both aliphatic and aromatic amino acids was examined in acetonitrile containing 70% (v/v) of 0.02 M phosphate buffer (pH 7.0) at 30°C. With all amino acids tested, the corresponding l-enantiomers were hydrolyzed preferentially. PPL favored aromatic amino acids, such as phenylalanine and p-chlorophenylalanine, leading to completion of the hydrolysis within 20 min with excellent enantioselectivities (E>100). The PPL-catalyzed hydrolysis of the corresponding Cam esters (Z-dl-AA-OCams) was also examined under the same reaction conditions. Although the hydrolysis of the Cam esters was rapid, the l-enantioselectivities were rather poor with aromatic amino acids, such as 2-phenylglycine and homophenylalanine.     

The influence of structural components of alkyl esters on their anaerobic biodegradation in marine sediment

Ester-based organic compounds are one type of synthetic base fluid added to drilling mud used during off-shore oil-drilling operations in the Gulf of Mexico. Concern over the environmental impact of synthetic base fluid (SBF) contaminated rock cuttings discharged into the Gulf of Mexico has prompted the promulgation of EPA regulations requiring that all SBF be tested for biodegradability in marine sediment prior to their use in the Gulf. In order to allow the design or selection of suitably biodegradable esters, the anaerobic biodegradability of a variety of ester compounds was tested using a marine sediment inoculum to reveal the effect of: (a) increasing the chain length of the acid moiety, (b) increasing the chain length of the alcohol moiety; (c) alternating the relative size of the alcohol and acid moieties, (d) branching in the alcohol moiety, and (e) the presence of an unsaturated bond in the acidic moiety. The chemical structure of esters was found to affect the completeness and rate of anaerobic biodegradation, and would affect their ability to be certified for use as an SBF in the Gulf of Mexico. Recommendations for ester usage include using esters that have a total carbon number of between 12 and 18 and avoiding the use of branched alcohols (or acids by inference). The presence of an unsaturated bond in the acid (or alcohol by inference) increased biodegradability of the ester.     

Chloroplast Replication in Euglena gracilis Grown in Cadmium-Ion Containing Media

N-(1-Alkenyl)-2-chIoroacetamides, which bear an aryl substituent at the 1-position of the alkenyl moiety, were synthesized and their herbicidal activities were tested. Some of them were active against paddy field weeds. In particular, a new type of chloroacetamide herbicide, 2-chloro-N-methyl-N-(2-methyl-1-phenylpropen-1-yl)acetamide, having no 2,6-dialkyl substituted phenyl moiety, which has been considered essential for activity, was found to be active against barnyardgrass.     

Rapid ester biosynthesis screening reveals a high activity alcohol‐O‐acyltransferase (AATase) from tomato fruit

Ethyl and acetate esters are naturally produced in various yeasts, plants, and bacteria. The biosynthetic pathways that produce these esters share a common reaction step, the condensation of acetyl/acyl‐CoA with an alcohol by alcohol‐O‐acetyl/acyltransferase (AATase). Recent metabolic engineering efforts exploit AATase activity to produce fatty acid ethyl esters as potential diesel fuel replacements as well as short‐ and medium‐chain volatile esters as fragrance and flavor compounds. These efforts have been limited by the lack of a rapid screen to quantify ester biosynthesis. Enzyme engineering efforts have also been limited by the lack of a high throughput screen for AATase activity. Here, we developed a high throughput assay for AATase activity and used this assay to discover a high activity AATase from tomato fruit, Solanum lycopersicum (Atf‐S.l). Atf1‐S.l exhibited broad specificity towards acyl‐CoAs with chain length from C₄ to C₁₀ and was specific towards 1‐pentanol. The AATase screen also revealed new acyl‐CoA substrate specificities for Atf1, Atf2, Eht1, and Eeb1 from Saccharomyces cerevisiae, and Atf‐C.m from melon fruit, Cucumis melo, thus increasing the pool of characterized AATases that can be used in ester biosynthesis of ester‐based fragrance and flavor compounds as well as fatty acid ethyl ester biofuels.     

Enhanced Resolution of a Secondary Alcohol by Hydrolysis of a Bichiral Ester Catalyzed by Lipase from Candida cylindracea

The effect of a chiral centre in the acyl group on the resolution of esters prepared from a racemic alcohol was investigated. R-2-chloropropionic acid afforded a higher enantiomeric ratio than S-2-chioropropionic acid in the hydrolysis of the corresponding esters of racemic 1-phenylethanol catalyzed by Candida cylindracea lipase. Even when a mixture of esters prepared from racemic acid and racemic alcohol was used for resolution of the alcohol, a noteworthy high enantioselectivity was observed. The hydrolysis of a bichiral ester offers an amplification in the resolution of enantiomers of alcohols by the combination of a chemical diastereoselectivity and an enzymatic enantio- and diastereoselectivity.     

Hydrolytic resolution of (R,S)-2-hydroxycarboxylic acid esters in biphasic media: implication for rate-limiting formation or breakdown of tetrahedral intermediates in acylation step

A thermally stable esterase (SNSM‐87) from Klebsiella oxytoca is explored as an enantioselective biocatalyst for the hydrolytic resolution of (R,S)‐2‐hydroxycarboxylic acid esters in biphasic media, where the best methyl esters possessing the highest enantioselectivity and reactivity are selected and elucidated in terms of the structure–enantioselectivity correlations and substrate partitioning in the aqueous phase. With (R,S)‐2‐chloromandelates as the model substrates, an expanded Michaelis–Menten mechanism for the rate‐limiting acylation step is adopted for the kinetic analysis. The Brønsted slope of 25.7 for the fast‐reacting (S)‐2‐chloromandelates containing a difficult leaving alcohol moiety, as well as that of 4.13 for the slow‐reacting (R)‐2‐chloromandelates in the whole range of leaving alcohol moieties, indicates that the breakdown of tetrahedral intermediates to acyl‐enzyme intermediates is rate‐limiting. However, the rate‐limiting step shifts to the formation of tetrahedral intermediates for the (S)‐2‐chloromandelates containing an easy leaving alcohol moiety, and leads to an optimal enantioselectivity for the methyl ester substrate. Biotechnol. Bioeng. 2007; 98: 30–38. © 2007 Wiley Periodicals, Inc.     

Structure-activity relationships of bergenin derivatives effect on α-glucosidase inhibition

The α-glucosidase inhibitory activities of bergenin derivatives were evaluated. Bergenin derivatives were synthesized from bergenin which is a characteristic compound of B. ligulata. A new bergenin derivative, 11-O-(3′,4′-dimethoxybenzoyl)-bergenin showed the highest potent inhibitory activity among those of bergenin derivatives. The presence of substituents at 3′,4′-position in bergenin derivatives altered the α-glucosidase inhibitory activity. 11-O-(3′,4′-dimethoxybenzoyl)-bergenin was noncompetitive inhibitor for α-glucosidase. The present study reveals that bergenin derivatives could be classified as a new group of α-glucosidase inhibitors.