Lactones 29 . Enzymatic resolution of racemic γ-lactones

Studies on the application of commercially available enzymes to resolution of the racemic unsaturated γ-lactones: 5-(3-methylbutylidene)-4-methyl-tetrahydrofuran-2-one (1a) and 5-(3,3-dimethylbutylidene)-4-methyl-tetrahydrofuran-2-one (2a) are presented. Lipase PS, Rhizopus niveus lipase, Rhizopus arrhizus lipase, porcine pancreas lipase and hog liver esterase transformed substrates into their respective γ-keto acids with good efficiency (50-75%). Three of them hydrolysed the studied lactones with moderate enantioselectivity. Enantiomeric excesses determined by GC for the unreacted lactones were in the range of 20-60%. Lipase PS preferentially hydrolysed the (+) enantiomers of lactones 1a and 2a whereas R. niveus lipase hydrolysed the (-) enantiomers, respectively.     

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.     

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.     

Lipase-catalyzed optical resolution of trifluoro(aryl)ethanols

Optical resolutions of racemic 2,2,2-trifluoro-1-(aryl)ethanols — (1-naphthyl), (2-naphthyl), (4-methylnaphthyl), (phenyl), (1-pyrenyl) — were achieved by lipase-catalyzed enantioselective acetylations with vinyl acetate as an acetyl donor in octane, and (S)-acetates and (R)-alcohols were obtained. Among the lipases tested, lipase from Pseudomonas aeruginosa (lipase LIP, Toyobo) showed good enantioselectivity for above ethanols. However, no acetylation occurred with sterically hindered alcohols — (9-phenanthryl), (9-anthryl), (2-methylnaphthyl), (2, 4, 6-trimethylphenyl) — by various lipases. The resolutions of the three alcohols were carried out by the enantioselective alcoholysis or hydrolysis of their chloroacetates by lipase LIP.     

定点突变提高环氧化物水解酶AuEH2催化对甲基苯基缩水甘油醚的对映选择性*

环氧化物水解酶可催化外消旋环氧化物的动力学拆分或对映归一性水解制备手性环氧化物或邻二醇,具有广阔的应用前景.为提高宇佐美曲霉环氧化物水解酶 (AuEH2) 催化外消旋对甲基苯基缩水甘油醚 (rac-pMPGE) 的对映体选择率 (E).通过分子动力学模拟 (MD) 选取相互作用频率最高的位点A250替换为其他19种氨基酸;选取对映选择性显著提高的突变体测定其动力学参数 (K_m和k_cat) 及区域选择性系数 (β_S和β_R),并利用重组大肠杆菌全细胞拆分rac-pMPGE.突变体AuEH2^A250H的E值从12.7提高至38.4,重组菌比活力为51.9U/g湿细胞;其水解 (S)-pMPGE的k_cat/K_m从10.0mmol/(L·s)提高至12.8 mmol/(L·s),而水解 (R)-pMPGE的k_cat/K_m从1.13mmol/(L·s)降低至0.35mmol/(L·s);全细胞拆分20mmol/L rac-pMPGE获得 (R)-pMPGE的ee_s为>99%,产率从33.0% 提高至40.7%.A250位点的突变对AuEH2的对映选择性和酶活力具有显著影响;高对映选择性的AuEH2突变体在制备高光学纯的 (R)-pMPGE中具有应用潜力.     

Overexpression of Serratia marcescens lipase in Escherichia coli for efficient bioresolution of racemic ketoprofen

Lipase from Serratia marcescens ECU1010 was cloned and overexpressed in E. coli. After optimization, the maximum lipase activities reached 5000–6000 U/l and this recombinant lipase could enantioselectively hydrolyze (S)-ketoprofen esters into (S)-ketoprofen. Among six alkyl esters of racemic ketoprofen investigated, this lipase showed the best enantioselectivity for the kinetic resolution of ketoprofen ethyl ester, with an ee_p (enantiomeric excess of product) of 91.6% and E-value of 63 obtained at 48.2% conversion. Twelve nonionic surfactants were tested for enhancing the enantioselectivity of this lipase in the bioresolution of ketoprofen ethyl ester. A very high E-value of 1084 was achieved, with an optical purity of >99% ee_p and a yield of 42.6% in the presence of 3% Brij 92V. Further studies showed that the selectivity of the lipase was improved with the increase of Brij 92V concentration. The substrate (ketoprofen ethyl ester) does not inhibit the lipase activity, while the product (S)-ketoprofen inhibits the lipase activity to some extent. These results indicate that the S. marcescens lipase is very useful for biocatalytic production of chiral profens such as (S)-ketoprofen.     

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.     

Kinetic and modelling studies on the lipase catalysed enantioselective esterification of (±)-perillyl alcohol

Several lipases were kinetically studied with the aim to exploit their enantioselectivity in the esterification of (S)-(−) and (R)-(+)-perillyl alcohol with decanoic acid. Most of the lipases studied exhibited stereopreference towards the R-enantiomer with apparent E-values from 3.8 to 0.6, calculated as the initial esterification rates ratio for the individual enantiomers. In an attempt to interpret the structural basis of enantioselectivity, modelling studies were performed with two of these lipases, Candida cylindracea lipase (CcL) and Pseudomonas cepacia lipase (PcL) based on their previously determined X-ray crystal structures. The results derived from modelling studies confirm their stereopreferences towards the R-enantiomer, since increased conformational energy of the S-ester was found compared to the R-ester.     

Preparation of both enantiomers of methyl 3-benzoyloxypentanoate by enzyme-catalysed hydrolysis of corresponding racemic nitrile and amide

Rhodococcus rhodochrous IFO 15564 enantioselectively hydrolysed racemic 3-benzoyloxypentanenitrile and 3-benzoyloxypentanamide to afford (R)-amide and (S)-car☐ylic acid with high enantiomeric excess (> 90%). In this reaction, both enantiomers of the starting nitrile were converted to the amide by nitrile hydratase, and amidase-catalysed enantioselective hydrolysis of the amide was responsible for the kinetic resolution. The lack of enantioselectivity of the nittile hydratase toward the racemic nitrile forms a marked contrast to the case of previously reported highly enantioselective conversion of prochiral 3-benzoyloxypentanedinitrile by this enzyme. since (R)-amide could be hydrolysed chemically to (R)-car☐ylic acid without any loss of its ee, the present microbial kinetic resolution serves as an effective method for preparing both enantiomers of synthetically useful 3-hydroxypentanoic acid derivatives.     

Microfluidic separation of (S)-ibuprofen using enzymatic reaction

This study was investigated for the enantioselective separation of (S)-ibuprofen using the ionic liquid in the microfluidic device. A stable and thin ionic liquid flow (ILF) was made by controlling the flow rate of the ILF in the microfluidic channel. In addition, coupling lipase as a biocatalyst with the ILF based on the microfluidic device showed the facilitative and selective transport of (S)-ibuprofen across the ILF, indicating successful optical resolution of a racemic mixture. Subsequently, the enantioselectivity was evaluated in the transport ratio (η) of (R)- and (S)-ibuprofen, the optical resolution ratio () and enantiomeric excess of (S)-ibuprofen (ee_S).     

Resolution of N-(2-ethyl-6-methylphenyl) alanine catalyzed by Lipase B from Candida antarctica

A biotransformation process has been developed for the production of (S)-N-(2-ethyl-6-methylphenyl) alanine by enantioselective hydrolysis of racemic methyl ester in the presence of Candida antarctica lipase B (CAL-B). However, the enantioselectivity of CAL-B towards the resolution is not high enough to obtain enantiomerically pure product. In order to improve the enantioselectivity of the enzyme, the effects of surfactants on CAL-B-catalyzed hydrolysis were tested. The results suggest that surfactants can influence the microenvironment of the enzyme, and the addition of Tween-80, in particular, to the reaction medium markedly enhanced the selectivity of CAL-B towards the substrate used, with the enantiomeric ratio (E-value) increasing from 11.3 to 60.1.     

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.     

Cloning and optimization of a nitrilase for the synthesis of (3S)-3-cyano-5-methyl hexanoic acid

In this paper we describe the cloning and optimization of a nitrilase for a regio- and stereo-specific synthesis of (3S)-3-cyano-5-methyl hexanoic acid (2) from isobutylsuccinonitrile (IBSN, 1). Ten representative plant and bacterial nitrilases have been cloned and their substrate specificity was studied using a fluorescent assay. The desired nitrilase AtNit1 from Arabidopsis thaliana was identified with high enantioselectivity (E > 150). This enzyme was then purified and characterized to be an oligomer of 12 subunits by size exclusion chromatography. AtNit1 was subsequently optimized to increase expression and engineered to improve activity. Preliminary screening of a small percentage (1%) of the mutant library shows that the mutant C236S has a nearly 3-fold increase in reactivity in the hydrolysis of IBSN.     

酵母脂肪酶分子进化及催化合成(S)-1,4-苯并二口恶烷*

Objective: 1,4-Benzodioxane is an important chiral intermediate for antihypertensive (Proroxan and Doxazosin), antidepressant (MCK-242) and other drugs, and it displays a broad spectrum of applications in the pharmaceutical field. Currently, in spite of high-yield advantage of chemical synthesis, there are some problems of environmental pollution and low production safety. Using lipase to catalyze synthesis of 1,4-benzodioxane provides a new pathway of green synthesis of 1,4-benzodioxane. However, natural enzymes face the dilemma of poor enantioselectivity. Therefore, molecular evolution was performed on Candida antarctica lipase B, and a technical route for the catalytic synthesis of 1,4-benzodioxane was established. Methods: Firstly, the key amino acid residues involved in substrate binding and conversion in the active center of Candida antarctica lipase B were analyzed, and saturation mutagenesis libraries on the interaction sites were constructed. Improved mutants with high efficiency and high enantioselectivity were then obtained using HPLC detection. Furthermore, catalytic synthesis conditions of mutant D223N/A225K were systematically optimized. Results: The results indicated that the mutants mainly derived from the pairwise site D223/A225 (such as D223N/A225K and D223G/A225W) were biased towards the synthesis of (S)-isoforms, while most of the mutants derived from the pairwise site E188/I189 (such as E188D/I189M) showed a bias for the synthesis of (R)-isoforms. Compared with WT, the ee_s value of the best mutant D223N/A225K to synthesize (S)-1,4-benzodioxane was increased from 11.9% to 29.3%. After systematic optimization of the reaction conditions, an ee_s value of (93.9±0.16)% and a conversion rate of (47.5±2.33)% were achieved using mutant D223N/A225K to catalyze kinetic resolution of methyl (R,S)-2,3-dihydro-1,4-benzodioxin-2-carboxylate in n-butanol/phosphate buffered saline (20∶80, V/V) biphasic solvent at 37℃ for 50 min. Conclusion: An efficient kinetic resolution of methyl (R,S)-2,3-dihydro-1,4-benzodioxin-2-carboxylate was successfully achieved by molecular evolution and optimization of conditions, which provides a new example for the creation of new enzymes by protein engineering technology, and also provides a theoretical and technical foundation for the efficient synthesis of (S)-1,4-benzodioxane molecules by enzymatic methods.     

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.     

Biotransformation of β-hydroxyphenyl selenides, diphenyldiselenide and benzeneseleninic acid by whole cells of Aspergillus terreus

Aspergillus terreus CCT 3320 and A. terreus URM 3571 catalysed the biotransformations of organic β-hydroxyphenyl selenides through oxidation and methylation reactions. The kinetic resolution of (RS)-1-(phenylseleno)-2-propanol (1) via enantioselective oxidation produced (+)-(S)-1 in high enantiomeric excess (>99%) and in a yield of 50% as determined by product isolation. Oxidation of the R-enantiomer of 1, followed by elimination of the propyl moiety and subsequent methylation of the presumed intermediate, led to the formation of methylphenyl-selenide, which was isolated in a yield of 40%. Whole cells of A. terreus also biocatalysed transformations of diphenyldiselenide, benzeneseleninic acid, (RS)-1-(phenylseleno)-2-pentanol and (RS)-1-(phenylseleno)-3-methyl-2-butanol, but not of (RS)-1-(phenylseleno)-2-phenyl-methanol. This is the first report of the biomethylation of organoselenium compounds by whole cells of A. terreus.     

Lipase-catalyzed enantioselective hydrolysis of methyl 2-fluoro-2-arylpropionates in water-saturated isooctane

Lipases from Candida rugosa, Candida antartica B and Carica papaya are employed as the biocatalyst for the hydrolytic resolution of methyl 2-fluoro-2-arylpropionates in water-saturated isooctane, in which excellent to good enantioselectivity without the formation of byproducts is obtained for the papaya lipase when using (R,S)-2-fluoronaproxen methyl ester (1) and methyl (R,S)-2-fluoro-2-(4-methoxyphenyl)propionate (2), but not methyl (R,S)-2-fluoro-2-(naphth-1-yl)propionate (3) as the substrates. The thermodynamic analysis indicates that the enantiomer discrimination for the papaya lipase is driven by the difference in activation enthalpy for compound 1, 2 or (R,S)-naproxen methyl ester (4). The kinetic analysis also demonstrates that in comparison with (S)-4, the insertion of the 2-fluorine moiety in (R)-1 has increased k₂, but not K_m, and consequently the lipase activity.     

Screening of lipases for regioselective hydrolysis of peracetylated β-monosaccharides

The monodeacetylation of peracetylated-β-d-galactose (1) and peracetylated N-acetyl-β-d-glucosamine (2) by different lipases is here described. Lipases from different sources in an immobilized form were evaluated to find those that offer the higher activity and regioselectivity in the reactions. In the hydrolysis of 1, the lipase from Aspergillus niger was the most active one, although it hydrolyzed the anomeric position. Using the lipase from Candida rugosa, 30% yield of the corresponding 6-OH isomer was achieved. On the other hand, in the hydrolysis of 2, the lipase from A. niger was the most active and regioselective catalyst, producing more than 75% of the 6-OH derivative product.     

Immobilized cells biocatalyst for the production of S-acetylthio-2-methyl propionic acid

S-acetylthio-2-methylpropionic acid (S-AMPA) is an important chiral intermediary for numerous hypertension drugs such as captopril. S-AMPA can be produced by hydrolyzing the corresponding racemic methyl MAMP (S,R-methyl-β-acetylthioisobutyrate) by lipases or esterases that have the appropriate stereo specificity. Psudomonas fluorescens IFO 12055 possessing a highly specific lipase was used to process this reaction in the form of immobilized cells. Reaction kinetic and immobilization methods were also studied. Strong product inhibition was observed, that is, at 3% S-AMPA (namely 183 mM), activity was reduced by 50%. Spontaneous hydrolysis of the ester and thioester bonds was also observed, and was independent of the cells. Thus, reaction selectivity and yield must be optimized through adjusting the substrate concentration and total biocatalyst activity. Conventional calcium alginate (3% w/w) encapsulation was modified by adding 3% w/w polyethyleneimine (PEI) and cross-linked by a biologically derived agent, genipin (5.6 mM). This method was found to be satisfactory to produce stable and functioning biocatalyst and can maintain high reactivity for repeated 25 batches with e.e. values above 90%.     

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.