Chemoenzymatic synthesis of four diastereomers of (6-fluoro-2-chromanyl) oxirane: An intermediate of a potent β-blocker

The synthesis of optically active 5-acetoxy-3-(p-fluorophenoxy)-1-pentanol 4, for the synthesis of the potent β-blocker R-67555, bis[2-(2-chromanyl-6-fluoro)-2-hydroxyethyl]amine 1, was investigated. The acetylation of 3-(p-fluorophenoxy)-1,5-pentanediol 5a using lipozyme and the hydrolysis of 1,5-diacetoxy-3-(p-fluorophenoxy)pentane 5b using lipase Amano P yielded (3S)- and (3R)-5-acetoxy-3-(p-fluorophenoxy)-1-pentanol 4, respectively, with high enantiomeric excess. Four diastereomers of (6-fluoro-2-chromanyl)oxirane 2, important intermediates for the synthesis of R-67555, were synthesized by chemical methods using (S)-4 and (R)-4.     

Lipase-catalyzed production of optically active (S)-flurbiprofen in aqueous phase reaction system containing chiral succinyl β-cyclodextrin

The lipase-catalyzed production of optically active (S)-flurbiprofen was carried out in a dispersion reaction-system induced by chiral succinyl β-cyclodextrin (suβ-CD). The optimal reaction conditions were 500 mM (R,S)-flurbiprofen ethyl ester ((R,S)-FEE), 600 units of Candida rugosa lipase per 1 mmol of (R,S)-FEE, and 1000 mM suβ-CD at 37 °C for 72 h. An extremely high enantiomeric excess of 0.98 and conversion yield of 0.48 were achieved in the dispersed aqueous phase reaction system containing chiral suβ-CD added as a dispenser and chiral selector. The inclusion complex formability of the immiscible substrate (S)- and (R)-form of FEE with suβ-CD was compared using a phase-solubility diagram, DSC, and ¹H NMR. (S)-Isomer formed a more stable and selective inclusion complex with chiral suβ-CD. It was hydrolyzed much more selectively by lipase from C. rugosa, due to the selective structural modification through inclusion complexation with chiral suβ-CD.     

Fed-batch production of (S)-flurbiprofen in lipase-catalyzed dispersed aqueous phase reaction system induced by succinyl β-cyclodextrin and its extractive purification

Optically active (S)-flurbiprofen was produced fed-batch-wisely in a lipase-catalyzed dispersed aqueous phase reaction system induced by succinyl β-cyclodextrin (suβ-CD). A highly concentrated 480 mM (S)-flurbiprofen, corresponding to 117.0 g/l, with an enantiomeric excess of 0.98 and conversion yield of 0.48 was obtained. (S)-Flurbiprofen produced in an inclusion complex form with suβ-CD was extractively purified using three-step procedures: decomplexation of (S)-flurbiprofen and residual (R)-flurbiprofen ethyl ester ((R)-FEE) using the ethyl acetate, dissolution of (S)-flurbiprofen from (R)-FEE using a sodium bicarbonate solution, and selective precipitation of (S)-flurbiprofen using 2-propanol. Consequently, an extremely high concentration of 420 mM (S)-flurbiprofen with an optical purity higher than 98% was recovered after purification.     

Oxidation of 1,4-alkanediols into γ-lactones via γ-lactols using Rhodococcus erythropolis as biocatalyst

Whole cells of Rhodococcus erythropolis DSM 44534 grown on ethanol, (R)- and (S)-1,2-propanediol were used for biotransformation of racemic 1,4-alkanediols into γ-lactones. The cells oxidized 1,4-decanediol (1a) and 1,4-nonanediol (2a) into the corresponding γ-lactones 5-hexyl-dihydro-2(3H)-furanone (γ-decalactone, 1c) and 5-pentyl-dihydro-2(3H)-furanone (γ-nonalactone, 2c), respectively, with an EE(R) of 40–75%. The transient formation of the γ-lactols 5-hexyl-tetrahydro-2-furanol (γ-decalactol, 1b) and 5-pentyl-tetrahydro-2-furanol (γ-nonalactol, 2b) as intermediates was observed by GC–MS. 1,4-Pentanediol (3a) was transformed into 5-methyl-dihydro-2(3H)-furanone (γ-valerolactone, 3c) whereas (R)- and (S)-2-methyl-1,4-butanediol (4a) was converted to the methyl-substituted γ-butyrolactones 4-methyl-dihydro-2(3H)-furanone (4c₁) and 3-methyl-dihydro-2(3H)-furanone (4c₂) in a ratio of 80:20 with a yield of 55%. Also cis-2-buten-1,4-diol (5a) was transformed resulting in the formation of 2(5H)-furanone (γ-crotonolactone, 5c). At the higher pH values of 8.8 the yield of lactone formed was improved; however, the enatiomeric excesses were slightly higher at the lower pH of 5.2.     

Novel antioxidant agents deriving from molecular combinations of vitamins C and E analogues: 3,4-dihydroxy-5(R)

Molecular combinations of two antioxidants (i.e., ascorbic acid and the pharmacophore of -tocopherol), namely the 2,3-dihydroxy-2,3-enono-1,4-lactone and the chromane residues, have been designed and tested for their radical scavenging activities. When evaluated for their capability to inhibit malondialdehyde (MDA) production in rat liver microsomal membranes, the 3,4-dihydroxy-5R-2(R,S)-(6-hydroxy-2,5,7,8-tetramethylchroman-2(R,S)yl-methyl)-1,3]dioxolan-4S-yl]-5H-furan-2-one (11a–d), exhibited an interesting activity. In particular the 5R,2R,2R,4S and 5R,2R,2S,4S isomers (11c,d) displayed a potent antioxidant effect compared to the respective synthetic -tocopherol analogue (5) and natural -tocopherol or ascorbic acid, used alone or in combination. Moreover, the mixture of stereoisomers 11a–d also proved to be effective in preventing damage induced by reperfusion on isolated rabbit heart, in particular at the higher concentration of 300 μM. In view of these results our study represents a new approach to potential therapeutic agents for applications in pathological events in which a free radical damage is involved. Design, synthesis and preliminary biological activity are discussed.     

The Production of γ-Decalactone by Fermentation of Castor Oil

A microbial process for the production of optically-active γ-decalactone from the ricinoleic acid present as triglycerides in castor oil has been developed, γ-decalactone (γDL) is a component of some fruit flavours, being an important organoleptic component of peach flavours. Screening showed two red yeast microorganisms, Rhodotorula glutinis and Sporobolomyces odortts to be especially suitable for this biotransformation. The process involves lipase-mediated hydrolysis of the castor oil to give free ricinoleic acid, uptake of the acid by the cells and aerobic fermentation to achieve abbreviated β-oxidation of the ricinoleic acid (12-hydroxyoleic acid) into 4-hydroxydecanoic acid (4HDA), lactonisation of the acid into γ-DL, followed by solvent extraction and distillation. γ-DL broth concentrations of 0.5-1.2g · 1^-t were obtained after 3-5 days from fermentation media containing 10 g · 1^-1 castor oil, representing an 8.3-20.0% theoretical yield. Intermediates detected were consistent with the operation of the β-oxidation pathway. Appreciable amounts of novel metabolites identified as cis and trans isomers of a tetrahydrofuran (C₁₀) were also produced. Their formation from 4HDA appeared to be non-enzymic and was favoured by anaerobic conditions. Yields of γ-DL were inversely proportional to the concentration of castor oil present in the medium, indicating that substrate inhibition takes place. The highest yields of γ-DL were obtained when castor oil was present from the beginning of the fermentation, rather than when added once the fermentation had become established, demonstrating that the β-oxidation pathway and/or transport system require continual induction. Significant amounts of γ-DL were not produced from other fatty acids, including ricinelaidic acid, the trans isomer of ricinoleic acid. γ-DL formation was dramatically inhibited by antibiotic inhibitors of oxidative phosphorylation, indicating the importance of intact β-oxidation pathways, whereas inhibitors of protein synthesis and cell-wall synthesis had much less marked effects. Selective extraction of 4HDA from the fermentation broths, and of γDL from broth lactonised by heating at low pH, could be achieved by adsorption to Amberlite XAD-1 and XAD-7 resins respectively. Some product could be recovered from the exit gases of the fermenter by passing through propylene glycol traps. This pathway is unusual in that it is a rare example of the truncated β-oxidation of a fatty acid by microorganisms. This effect probably occurs because of partial inhibition of one or more enzymes of the β-oxidation pathway by the C₁₀ hydroxylated fatty acid intermediate(s) allowing intracellular accumulation of the 4HDA, followed by leakage out of the cell; although further metabolism of this C₁₀ intermediate does take place slowly.     

Esterified, optical pure (3S, 3′S)-astaxanthin from flowers of Adonis annua

The quantitative carotenoid composition of the red flower petals of Adonis annua is reported. Optically pure (3S, 3′S)-astaxanthin occurs both as a diester (64% of total carotenoid) and as a monoester (11%). The optical purity was determined by hydrolysis of the natural esters in the absence of oxygen and subsequent HPLC analysis of the paren -ketol esterified with (−)-camphanic acid. All non-animal sources hitherto examined synthesize pure 3S,3′S- or 3R,3′R-isomers of astaxanthin, whereas marine animal sources contain mixtures of all three optical isomers, including the meso form.     

Generation and analyses of the transgenic potatoes expressing heterologous thermostable β-amylase

β-Amylase hydrolyzes the -1,4-glycosidic linkages of starch resulting in the release of maltose. This reaction is of industrial importance for maltose production and for the preparation process of fermented foods and alcoholic beverages. A demand for an acceleration of the rate of enzymatic cleavage of the starch macro-molecule is a prerequisite for large-scale and highly efficient production. Increasing the temperature up to the optimum of approximately 60 °C can significantly speed up the reaction. However, at higher temperatures, the effect on protein denaturation becomes dominant, and the conversion rate decreases. The primary objective of this study was to generate transgenic plants of the “Kennebec” potato variety for production of thermostable β-amylase using Agrobacterium-mediated transformation. Four chimeric genes encoding the β-amylase with or without signal peptide sequences for targeting expression in cytoplasm, amyloplasts, or vacuoles were constructed and driven by high tuber expression promoter from Sucrose synthetase gene Sus4. Forty-two transgenic lines were selected for this study. Transgenic lines with various β-amylase constructs were verified for the existence and expression of the transgenes by PCR approaches. The expression level of the introduced β-amylase protein was estimated by immunoblot analyses using polyclonal antibodies. Recombinant β-amylase was successfully expressed in Escherichia coli B21 (DE3), and temperature ranges of these inducible recombinant proteins were found to be between 40 and 90 °C. This enzymatic complex produced in the in vitro cultured microtubers and field-grown tubers from transgenic potatoes were proved to be stable and active at 60 °C. The relative activities of β-amylase in tubers of field-grown potatoes were compared, and the maximum increase was found with transgenic line #6A of the pSUS4-AMY construct which has an 11-fold greater increase than the untransformed “Kennebec”. Variations of the chemical compositions were found in the selected transgenic lines. Results of this study suggest the feasibility of utilizing thermostable β-amylase in transgenic potatoes for the starch-processing industries.     

Asymmetric bioreduction of racemic 5,6,7,8-tetrahydro-8-methyl-1,3-dimethoxynaphthalen-6-one to the corresponding chiral β-tetralols

The synthesis of racemic 5,6,7,8-tetrahydro-8-methyl-1,3-dimethoxynaphthalen-6-one 1 was performed and the bioreduction to the corresponding β-tetralols was studied with respect to the stereochemical course and optical purity of the products; in particular the 6S,8S enantiomer corresponding to the dimethyl derivative of the natural compound feroxidin was isolated. The biomass of: Aspergillus niger, Cladosporium cladosporioides, Candida lypolitica, Bacillus megatherium, Rhodotorula minuta, R. flava, R. rubra, Beauveria bassiana and Baker's yeast were used as biocatalysts. Relative and absolute configurations of the obtained β-tetralols were established by comparison with those of the natural feroxidin.     

Fungus β-glycosidases: immobilization and use in alkyl-β-glycoside synthesis

Production of β-glycosidases: β-xylosidase and β-glucosidase by the fungus Sclerotinia sclerotiorum was optimized in the presence of different carbon sources. Immobilization supports with different physico-chemical characteristics were evaluated for use in continuous reactors. Immobilization and activity yields were calculated. Among the adsorption on Duolite, Amberlite, Celite and DEAE-sepharose, and entrapment in polyacrylamide gel or reticulation using glutaraldehyde, highest yields were obtained when β-xylosidase was adsorbed on Duolite A 7 and when β-glucosidase was adsorbed on DEAE-sepharose.

Enzyme preparations from S. sclerotiorum cultures were used in a biphasic (alcohol/aqueous) medium for the synthesis of alkyl-glycosides by trans-glycosylation of sugars and long-chain alcohols. The synthesis was studied under different conditions with primary and secondary alcohols as substrates, in the presence of free or immobilized enzyme. Xylan and cellobiose were used for the synthesis of alkyl-xylosides and alkyl-glucosides, respectively. The majority of the immobilized preparations were unable to catalyze the synthesis of alkyl-glycosides.

Highest yields were obtained when using xylan and C₄–C₆-alcohols. The reaction produced alkyl-β-xyloside and alkyl-β-xylobioside, as confirmed by MS/MS. Up to 22 mM iso-amyl-xyloside and 14 mM iso-amyl-xylobioside were produced from iso-amyl alcohol and xylan.     

Lipase-catalyzed kinetic resolutions of racemic β- and γ-thiolactones

Several racemic β- and γ-thiolactones were synthesized and kinetic resolutions of them were executed using lipases. While a lipase from Pseudomonas cepacia (PCL) showed the highest enantioselectivity for (S)-form (>99% ee_S at 53% conversion, E > 100) in the kinetic resolution of racemic -methyl-β-propiothiolactone (rac-MPTL), it showed no hydrolysis activity in the kinetic resolution of -benzyl--methyl-β-propiothiolactone (rac-BMPTL), suggesting that the changes in the size of alkyl group from rac-MPTL to rac-BMPTL leads to lower hydrolysis activity and enantioselectivity. In contrast, racemic γ-butyrothiolactones were hydrolyzed by several lipases with low enantioselectivity, whereas a lipase from Candida antarctica (CAL) showed moderate enantioselectivity for (S)-form (>99% ee_S at 76% conversion, E = 11) in the kinetic resolution of racemic -methyl-γ-butyrothiolactone (rac-MBTL). Computer-aided molecular modeling was also performed to investigate the enantioselectivites and activities of PCL toward β-propiothiolactones. The computer modeling results suggest that the alkyl side chains of β-propiothiolactones and γ-butyrothiolactones interact with amino acid residues around hydrophobic crevice, which affects the activity of PCL.     

Algal carotenoids 52; secondary carotenoids of algae 3; carotenoids in a natural bloom of Euglena sanguinea

Quantitative carotenoid analysis of a natural bloom of Euglena sanguinea Ehrenberg revealed the presence of β,β-carotene (1% of total carotenoids), monoesters of adonirubin (3%), diesters of (3S, 3′R)-adonixanthin (13%), diesters of (3S, 3′S)-astaxanthin (75%), 19-monoester of (3R, 3′R, 6R)-loroxanthin (1%), (3R, 3′R)-diatoxanthin (6%), diadinoxanthin (1%) and neoxanthin (traces). The carotenoid content amounted to 0.7% of the dry wt. Methods employed included TLC, HPLC, VIS, MS, CD and H NMR (400 and 500 MHz). The high content of ketocarotenoids is characteristic of secondary carotenoids produced under stressed growth conditions. Previously secondary carotenoids were associated with green algae (Chlorophyceae), but have now been encountered in Euglenophyceae.     

Enzymatic production of γ-L-glutamyltaurine through the transpeptidation reaction of γ-glutamyltranspeptidase from Escherichia coli K-12

γ-L-Glutamyltaurine is a naturally occurring peptide and known to have several physiological functions in mammals. This paper describes a new method for the enzymatic production of γ-L-glutamyltaurine from L-glutamine and taurine through the transpeptidation reaction of γ-glutamyltranspeptidase (EC 2.3.2.2) of Escherichia coli K-12. The optimum conditions for the production of γ-L-glutamyltaurine were 200 mM L-glutamine, 200 mM taurine and 0.2 U/ml γ-glutamyltranspeptidase, pH 10, and 1-h incubation at 37°C. Forty-five mM γ-L-glutamyltaurine was obtained, the yield being 22.5%. γ-L-Glutamyltaurine was purified on Dowex 1 × 8 and C₁₈ columns, and identified by means of NMR and a polarimeter.     

Structure and physicochemical properties of barley non-starch polysaccharides—II. Alkaliextractable β-glucans and arabinoxylans

Three fractions containing hemicellulosic material were obtained by sequential extraction of barley residue (left after removal of water-extractable polysaccharides) with saturated barium hydroxide [Ba(OH)₂ fraction], distilled water [Ba(OH)₂/H₂O fraction], and 1 m sodium hydroxide [NaOH fraction]. The yields of the fractions were 1.6, 1.7, and 2.6% (w/w), respectively, of the dry barley grist. The Ba(OH)₂ fraction contained mainly arabinose and xylose, 35.8% and 60.9%, respectively. The Ba(OH)₂/H₂O fraction in addition to 26.7% Ara and 36.6% Xyl contained also 34.8% Glc. The NaOH fraction was composed of 14.2% Ara, 44.0% Xyl, and 40.9% Glc. The Ba(OH)₂/H₂O and NaOH extracts were further fractionated by stepwise (NH₄)₂SO₄ precipitation into several subfractions with varying amounts of β-glucans and arabinoxylans. β-Glucans in Ba(OH)₂/H₂O and NaOH fractions were characterized by high ratios of β-(1→4)/β-(1→3) linkages, large amounts of contiguously linked β-(1→4) segments, and high ratios of cellotriosyl/cellotetraosyl units. The alkali-extractable arabinoxylans, especially those NaOH-extractable, were characterized by a very low degree of substitution, high xylose/arabinose ratio, and a small content of doubly substituted xylose residues. Some populations of arabinoxylans displayed structural features that would enable them to self-associate or to interact with β-glucans.     

Regioselective enzymatic aminoacylation of lobucavir to give an intermediate for lobucavir prodrug

Synthesis of lobucavir prodrug, L-valine, [(1S,2R,3R)-3-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (BMS 233866), requires regioselective coupling of one of the two hydroxyl groups of lobucavir (BMS 180194) with valine. Either hydroxyl group of lobucavir could be selectively aminoacylated with valine by using enzymatic reactions. N-[(Phenylmethoxy)carbonyl]-L-valine, [(1R,2R,4S)-2-(2-amino-6-oxo-1H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester (3, 82.5% yield), was obtained by selective hydrolysis of N,N′-bis[(phenylmethoxy)carbonyl]bis[L-valine], O,O′-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester (1) with lipase M, and L-valine, [(1R,2R,4S)-2-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (4, 87% yield) was obtained by hydrolysis of bis[L-valine], O,O′-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester, dihydrochloride (2), with lipase from Candida cylindracea. The final intermediate for lobucavir prodrug, N-[(phenylmethoxy)carbonyl]-L-valine, [(1S,2R,4R)-3-(2-amino-6-oxo-1H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester (5), could be obtained by transesterification of lobucavir using ChiroCLEC™ BL (61% yield), or more selectively by using immobilized lipase from Pseudomonas cepacia (84% yield).     

Stereochemistry of the hydrogen abstraction from pyridoxamine phosphate catalyzed by alanine racemase of Bacillus stearothermophilus

Alanine racemase of Bacillus stearothermophilus catalyzes transamination as a side reaction. Stereospecificity for the hydrogen abstraction from C-4′ of pyridoxamine 5′-phosphate occurring in the latter half transamination was examined. Both apo-wild-type and apo-fragmentary alanine racemases abstracted approximately 20 and 80% of tritium from the stereospecifically-labeled (4′S)- and (4′R)-[4′-³H]PMP, respectively, in the presence of pyruvate. Alanine racemase catalyzes the abstraction of both 4′S- and 4′R-hydrogen like amino acid racemase with broad substrate specificity. However, R-isomer preference is a characteristic property of alanine racemase.     

Lactones 20 []: Biohydroxylation of saturated bicyclic γ-lactones with the substituted cyclohexane system

Three bicyclic saturated γ-lactones with various numbers of methyl groups at the cyclohexane ring were transformed into the corresponding hydroxylactones. The most effective biohydroxylations were performed by means of cultures Fusarium culmorum and Absidia cylindrospora. Biotransformations of lactones 4a and b afforded the hydroxylactones with the secondary hydroxy groups (5a and b), whereas the hydroxy group introduced into the lactone 4c turned out to be tertiary giving the product 5c.     

Biotransformation of β-amino nitriles: the role of the N-protecting group

N-Tolylsulfonyl- and N-butyloxycarbonyl-protected β-amino nitriles were prepared to study the effect of the N-protecting group on the biotransformation of the β-amino nitriles to the corresponding β-amino amides and acids. The bioconversions were carried out by using whole cells of Rhodococcus sp. R312 and Rhodococcus erythropolis NCIMB 11540. The bioconversion products of five-membered carbocyclic nitriles were mainly the respective acids whereas the carbocyclic six-membered nitriles were accumulated at the stage of the amide. Benefits of the enzymatic compared with the chemical hydrolysis of β-amino nitriles are the mild reaction conditions for the transformation of the nitrile group in the presence of acid or base labile N-protecting groups. In the present work we concentrated on this chemoselectivity of the biotransformation rather than its potential enantioselectivity, which will be subject of future investigations. Thus, some new compounds were prepared: (±)-(2-cyano-cyclohexyl) carbamic acid tert-butyl ester (4a), (±)-(2-carbamoyl-cyclopentyl) carbamic acid tert-butyl ester (3b) and (±)-(2-carbamoyl-cyclohexyl) carbamic acid tert-butyl ester (4b).     

Thermostable β-Glycosidase from Sulfolobus Solfataricus

The Sulfolobus solfataricus β-glycosidase (Sβgly) is a thermostable and thermophilic glycosyl-hydrolase with broad substrate specificity. The enzyme hydrolizes β-D-gluco-, fuco-, and galactosides, and a large number of /Winked glycoside dimers and oligomers, linked β1-3, β1-4, and β1-6, It is able to hydrolize oligosaccharides with up to 5 glucose residues. Furthermore, it is also able to promote transglycosylation reactions. The corresponding gene has been cloned and overexpressed both in yeast and Escherichia coli. Based on sequence and functional data, the Sβgly has been assigned to the so-called BGA family of glycosyl-hydrolases, including β-glycosidases, β-galactosidases and phosho-β-galactosidases from mesophilic and thermophilic organisms of the three domains. The Sβgly has been crystallized and the resolution of its structure is in progress. Because of its special properties, the enzymes has considerable biotechnological potential.