Optimization of enantioselective resolution of racemic ibuprofen by native lipase from Aspergillus niger

Resolution of (R,S)-ibuprofen (2-(4-isobutylphenyl)propionic acid) enantiomers by esterification reaction with 1-propanol in different organic solvents was studied using native Aspergillus niger lipase. The main variables controlling the process (enzyme concentration and 1-propanol:ibuprofen molar ratio) have been optimized using response surface methodology based on a five-level, two-variable central composite rotatable design, in which the selected objective function was enantioselectivity. This enzyme preparation showed preferentially catalyzes the esterification of R(−)-ibuprofen, and under optimum conditions (7% w/v of enzyme and molar ratio of 2.41:1) the enantiomeric excess of active S(+)-ibuprofen and total conversion values were 79.1 and 48.0%, respectively, and the E-value was 32, after 168 h of reaction in isooctane.     

Hydroxynitrile lyase catalyzed cyanohydrin synthesis at high pH-values

The application of unusual high pH-values within enzymatic cyanohydrin synthesis has been investigated. Usually enzymatic cyanohydrin synthesis in two-phase systems requires low pH-values within the aqueous phase to suppress the non-enzymatic side reaction. In contrast, we investigated the usage of pH-values above pH 6 by using the highly enantioselective (S)-selective hydroxynitrile lyase from Manihot esculenta. With these unusual reaction conditions also the unfavorable substrate 3-phenoxy-benzaldehyde can be converted by the wild type enzyme with excellent conversion and enantiomeric excess yielding pure (S)-3-phenoxy-benzaldehyde cyanohydrin with an enantiomeric excess of 97%. Although the variant MeHNL–W128A shows a higher activity with respect to this reaction, the enantioselectivity was reduced (85% e.e.(S)). Additionally, a new continuous spectroscopic cyanohydrin assay monitoring the formation of 3-phenoxy-benzaldehyde cyanohydrin was developed. Dedicated to Prof. Dr. Christian Wandrey on the occasion of his 65th birthday.     

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 (α) and enantiomeric excess of (S)-ibuprofen (ee_S).     

AN EFFICIENT SYSTEM FOR THE ASYMMETRIC ACYLATION OF (R,S)-3-n-BUTYLPHTHALIDE CATALYZED BY NOVOZYME 435

Novozyme 435 could be a highly efficient catalyst in the asymmetric acylation of (R,S)-3-n-butylphthalide in tetrahydrofuran–hexane solvents. The effect of various reaction parameters such as agitation velocity, water content, mixed media, temperature, concentration of Novozyme 435, molar ratio of acetic anhydride to (R,S)-3-n-butylphthalide, reaction time, enantiomeric excess of substrate (ee_S), enantiomeric excess of product (ee_P), and enantioselective ratio (E) were studied. Tetrahydrofuran markedly improved (R,S)-3-n-butylphthalide conversion, enantiomeric excess of remaining 3-n-butylphthalide, and enantiomeric ratio. The optimum media were 50% (v/v) tetrahydrofuran and 50% (v/v) hexane. Other ideal reaction conditions were an agitation velocity of 150 rpm, 0.4% (v/v) water content, temperature of 30°C, 8 mg/mL dosage of Novozyme 435, 8:1 (0.4 mmol: 0.05 mmol) molar ratio of acetic anhydride to (R,S)-3-n-butylphthalide, and a reaction time of 48 hr. Under the optimum conditions, 96.4% ee_S and 49.3% conversion of (R,S)-3-n-butylphthalide were achieved. In addition, enantiomeric excess of the product was above 98.0%.     

Commercially viable resolution of ibuprofen

Ibuprofen belongs to the non-steroidal anti-inflammatory drug (NSAID) family known as profens. Studies demonstrate that (S-ibuprofen is 160 times more potent than (R-ibuprofen in vitro, while the accumulation of (R-ibuprofen can cause serious side effects such as gastrointestinal pain. Candida rugosa lipase was used to enantioselectively esterify racemic ibuprofen with decan-1-ol and butan-1-ol in cyclohexane with an enantiomeric ratio (E) of 130 and 46, respectively, in up to 46% conversion. Separation by bulb-to-bulb distillation of (R)-ibuprofen and unreacted alcohol from the corresponding (S)-alkyl ibuprofen ester was possible for the decyl but not the butyl case. The enantioselective hydrolysis of (S)-alkyl ibuprofen esters with the same biocatalyst in aqueous phosphate buffer was twice as slow for the decyl alcohol versus the butyl example. The combined environmentally friendly enantioselective esterification and hydrolysis of ibuprofen insured the isolation of (S)-ibuprofen with a greater than 99% enantiomeric excess.     

Microbial production of (S)-1-phenyl-1,3-propanediol by stereospecific reduction of 3-hydroxy-1-phenylpropane-1-one

A novel microbial method of synthesizing (S)-1-phenyl-1,3-propanediol [(S)-PPD] was developed in this study. Our laboratory stock cultures were screened for microorganisms that stereospecifically produced (S)-PPD from 3-hydroxy-1-phenylpropane-1-one (HPPO) using an intact cell system. Of the 828 strains examined (321 bacteria, 233 yeasts and 274 molds), certain strains of Williopsis saturnus var. mrakii and Cryptococcus albidus were found to produce (S)-PPD with over 99% enantiomeric excess (e.e.). Screening identified W. saturnus var. mrakii AJ-5620 as the most productive strain, and this strain was used for further experiments. The (S)-PPD-producing reaction using intact W. saturnus var. mrakii AJ-5620 cells was carried out by successive feeding of HPPO. A total (S)-PPD yield of 9.9 g/l was produced in 20 h. The molar yield was 81% and the optical purity of the (S)-PPD produced was over 99% e.e.     

Coexpression of a carbonyl reductase and glucose 6-phosphate dehydrogenase in Pichia pastoris improves the production of (S)-1-phenyl-1,2-ethanediol

Abstract

To develop an efficient biocatalyst to produce optically active (S)-phenyl ethanediol (PED), a carbonyl reductase SCRII and glucose 6-phosphate dehydrogenase were coexpressed intracellularly in Pichia pastoris. The recombinant enzyme PpSCRII was purified with a specific activity of 8.32 U mg^?1, over 36% higher than that of Escherichia coli SCRII. The recombinant cells P. pastoris/SCRIIG catalyzed the reduction of 2-hydroxyacetophenone to give (S)-PED with optical purity of >99% in a yield of 96.3%. The yield was improved by 19.9% and 25.7% over E. coli BL21/SCRII and Candida parapsilosis, respectively, when the reaction duration was shorted from 48 h to 24 h. When using glucose 50 g L^?1 as co-substrate, these P. pastoris/SCRIIG cells could be reused ten times and the optical purity and yield of (S)-PED kept at >99% enantiomeric excess and >85%, respectively.     

Site-Specific and Asymmetric Hydrolysis of Prochiral 2-Phenyl-1,3-propanediol Diacetate by a Bacterial Esterase from an Isolated Strain

Bacillus cereus 809A and Burkholderia sp. 711C were isolated from soil. These strains demonstrate hydrolysis activity towards prochiral 2-phenyl-1,3-propanediol diacetate and accumulated the corresponding chiral monoacetates into the reaction mixture. When 2-phenyl 1,3-propanediol diacetate was used as a substrate, the produced monoacetates with Burkholderia sp. 711C were obtained in a racemic form but that produced by Bacillus cereus 809A showed an excess of the (S)-form. The resting cell reaction revealed that for Bacillus cereus 809A, there was an enrichment of one of the enantiomers of the monoacetate such that the enantiomeric excess (e.e.) of the (S)-form was over 95%. The purified enzyme from Bacillus cereus 809A hydrolyzed diacetate to monoacetate, and the e.e. value of the (S)-form increased by prolonged reaction in a way similar to the resting cell reaction. From N-terminal amino acids, this esterase is conserved in some strains of Bacillus for which the genomic sequences have been reported.     

Highly enantioselective bioreduction of N-methyl-3-oxo-3-(thiophen-2-yl) propanamide for the production of (S)-duloxetine

Whole cells of Rhodotorula glutinis reduced N-methyl-3-oxo-3-(thiophen-2-yl) propanamide at 30 g/l to (S)-N-methyl-3-hydroxy-3-(2-thienyl) propionamide, an intermediate in the production of (S)-duloxetine, a blockbuster antidepressant drug, in 48 h. The reaction had excellent enantioselectivity (single enantiomer, >99.5% enantiomeric excess [ee]) with a >95% conversion.     

Production of enantiomerically pure (S)-phenyl(pyridin-2-yl)methanol with Lactobacillus paracasei BD101

Abstract

Asymmetric reduction studies of heteroaryl ketones, including phenyl(pyridin-2-yl)methanone in enantioselective form with biocatalysts are very few, and chiral heteroaryl alcohols have been synthesized generally in the small scale. In this study, seven bacterial strains have been used to produce the (S)-phenyl(pyridin-2-yl)methanol in high enantiomeric excess and yield. Among the tested strains, Lactobacillus paracasei BD101, was found to be the best biocatalyst for the reducing phenyl(pyridin-2-yl)methanone to the (S)-phenyl(pyridin-2-yl)methanol at gram scale. The asymmetric bioreduction conditions were systematically optimized using L. paracasei BD101, which demonstrated excellent enantioselectivity and high level of conversion for the bioreduction reaction. (S)-phenyl(pyridin-2-yl)methanol, which is an analgesic, was produced enantiomerically pure form in the first time on gram scale using a biocatalyst. In total, 5.857?g of (S)-phenyl(pyridin-2-yl)methanol in enantiomerically pure form (>99% enantiomeric excess) was obtained in 52?h with 93% yield using whole cells of L. paracasei BD101. Enantiomerically pure (S)-phenyl (pyridin-2-yl)methanol, which is an analgesic, was first produced in the gram scale using a biocatalyst with excellent ee (>99%) and yield (93%).     

Whole cell application of Lactobacillus paracasei BD101 to produce enantiomerically pure (S)‐cyclohexyl(phenyl)methanol

In this study, a total of 10 bacterial strains were screened for their ability to reduce cyclohexyl(phenyl)methanone 1 to its corresponding alcohol. Among these strains, Lactobacillus paracasei BD101 was found to be the most successful biocatalyst to reduce the ketones to the corresponding alcohols. The reaction conditions were systematically optimized for the reducing agent L paracasei BD101, which showed high enantioselectivity and conversion for the bioreduction. The preparative scale asymmetric reduction of cyclohexyl(phenyl)methanone ( 1 ) by L paracasei BD101 gave (S)‐cyclohexyl(phenyl)methanol ( 2 ) with 92% yield and >99% enantiomeric excess. The preparative scale study was carried out, and a total of 5.602 g of (S)‐cyclohexyl(phenyl)methanol in high enantiomerically pure form (>99% enantiomeric excess) was produced. L paracasei BD101 has been shown to be an important biocatalyst in asymmetric reduction of bulky substrates. This study demonstrates the first example of the effective synthesis of (S)‐cyclohexyl(phenyl)methanol by the L paracasei BD101 as a biocatalyst in preparative scale.     

Asymmetric reduction of (<Emphasis Type="Italic">S</Emphasis>)-3-chloro-1-phenylpropanol from 3-chloropropiophenone by preheated immobilized <Emphasis Type="Italic">Candida</Emphasis> <Emphasis Type="Italic">utilis</Emphasis>

An efficient method for asymmetric reduction of (S)-3-chloro-1-phenylpropanol from 3-chloropropiophenone was developed using preheated Candida utilis cells immobilized in calcium alginate gel beads. Heating the immobilized cells (bead diameter 1.5 mm) at 45°C for 50 min allowed the reaction to proceed with 99.5% enantiomeric excess (ee) and an 85% yield with 1 g substrate l⁻¹ (batch addition in three aliquots) in 48 h. The immobilized cells retained approximately 50% of their original catalytic activity after being reused three times.     

Production of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate using carbonyl reductase coupled with glucose dehydrogenase with high space–time yield

tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is an important chiral intermediate for the synthesis of rosuvastatin. The biotechnological production of (3R,5S)-CDHH is catalyzed from tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH) by a carbonyl reductase, and this synthetic pathway is becoming a primary route for (3R,5S)-CDHH production due to its high enantioselectivity, mild reaction conditions, low cost, process safety, and environmental friendship. However, the requirement of the pyridine nucleotide cofactors, reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) limits its economic flexibility. In the present study, a recombinant Escherichia coli strain harboring carbonyl reductase R9M and glucose dehydrogenase (GDH) was constructed with high carbonyl reduction activity and cofactor regeneration efficiency. The recombinant E. coli cells were applied for the efficient production of (3R,5S)-CDHH with a substrate conversion of 98.8%, a yield of 95.6% and an enantiomeric excess (e.e.) of >99.0% under 350 g/L of (S)-CHOH after 12 hr reaction. A substrate fed-batch strategy was further employed to increase the substrate concentration to 400 g/L resulting in an enhanced product yield to 98.5% after 12 hr reaction in a 1 L bioreactor. Meanwhile, the space–time yield was 1,182.3 g L⁻¹ day⁻¹, which was the highest value ever reported by a coupled system of carbonyl reductase and glucose dehydrogenase.     

Continuous production of chiral 1,3-butanediol using immobilized biocatalysts in a packed bed reactor: promising biocatalysis method with an asymmetric hydrogen-transfer bioreduction

An asymmetric hydrogen-transfer biocatalyst consisting of mutated Rhodococcus phenylacetaldehyde reductase (PAR) or Leifsonia alcohol dehydrogenase (LSADH) was applied for some water-soluble ketone substrates. Among them, 4-hydroxy-2-butanone was reduced to (S)/(R)-1,3-butanediol, a useful intermediate for pharmaceuticals, with a high yield and stereoselectivity. Intact Escherichia coli cells overexpressing mutated PAR (Sar268) or LSADH were directly immobilized with polyethyleneimine or 1,6-diaminehexane and glutaraldehyde and evaluated in a batch reaction. This system produced (S)-1,3-butanediol [87% enantiomeric excess (e.e.)] with a space time yield (STY) of 12.5 mg h⁻¹ ml⁻¹ catalyst or (R)-1,3-butanediol (99% e.e.) with an STY of 60.3 mg h⁻¹ ml⁻¹ catalyst, respectively. The immobilized cells in a packed bed reactor continuously produced (R)-1,3-butanediol with a yield of 99% (about 49.5 g/l) from 5% (w/v) 4-hydroxy-2-butanoate over 500 h.     

Chemoenzymatic resolution of racemic Wieland–Miescher and Hajos–Parrish ketones

Enantiospecific microbial reduction of bicyclic ketones was described. Racemic Wieland–Miescher (1) and Hajos–Parrish (2) ketones were used as substrates. In a 4-h biotransformation of Hajos–Parrish ketone (2) using the strain of Didymosphaeria igniaria an optically pure ketone (R)-2 was obtained, whereas the (S)-2 ketone underwent reduction to (4aS,5S)-4 alcohol with 100% of enantiomeric excess and with over 60% of diastereoisomeric excess. Jones oxidation of the alcohol obtained in the biotransformation gave an optically pure ketone (S)-2. Enzymatic system of Coryneum betulinum reduced the (R)-2 ketone to (4aR,5S)-4 alcohol with a high enantiomerical purity in a 6-day reaction. Wieland-Miescher (1) ketone was transformed by these microorganisms in an analogous way, but the reaction times were longer.     

Improving performance of Yarrowia lipolytica lipase lip2-catalyzed kinetic resolution of (R,S)-1-phenylethanol by solvent engineering

Extracellular Yarrowia lipolytica lipase Lip2 (YLIP2) demonstrated an (R)-enantiopreference for efficient resolution of (R,S)-1-phenylethanol by solvent engineering with different kinds of binary solvent. The enantioselectivity was significantly improved by the addition of 1, 4-dioxane. The reaction parameters including co-solvent concentration, reaction temperature, and the reaction time were optimized. When the reaction was carried out with n-hexane in the presence of 0.8% 1,4-dioxane at 50°C for 72 h, the enantiomeric excess of product markedly increased to 99.1% from 66% in pure n-hexane; the enantiomeric ratio was higher than 200, which was 500-fold compared with that in pure n-hexane. The results indicated that it is very important to design the proper co-solvents, especially to create appropriate micro-environment for YLIP2 for catalyzing the resolution of (R,S)-1-phenylethanol.     

Use of lipases in the resolution of racemic ibuprofen

Summary Resolution of (R,S)-ibuprofen enantiomers by esterification in different organic solvents was studied using Candida cylindracea lipase. This enzyme preparation had high enantiospecificity for S(+)-ibuprofen in the esterification reaction of a racemic ibuprofen with primary alcohols. The esterification yields of secondary alcohols were much lower than those of primary alcohols. Esterification with tertiary alcohols was not observed. The synthesis of esters was profoundly affected by the amount of water in the reaction mixture. C. cylindracea lipase was active only in very hydrophobic solvents. The esterification activity of the lipase was reduced significantly by addition of water. The R- and S-enantiomers of ibuprofen were determined without derivatization by HPLC using a chiral column.     

Asymmetric Hydrolysis of (±)-1-Acetoxy-2,3-dichloropropane with Enzymes and Microorganisms

Enzymes and microorganisms were screened for the enantioselective hydrolysis of (±)-1-acetoxy-2,3-dichloropropane (1) which is convertible to epichlorohydrin. Pancreatin and steapsin from hog pancreas were found to hydrolyze (±)-1 asymmetrically to give (S)-1 of 90% enantiomeric excess (e.e.). From (S)-1 was synthesized the optically pure (S)-isomer of propranolol[1-isopropylamino-3-(1-naphthoxy)-2-propanol], one of the typical β-adrenergic blocking agents.     

Enzyme catalyzed hydrolysis of the diesters of cis- and trans-cyclohexanedicarboxylic acids

Summary Pig liver esterase (EC 3.1.1.1) catalyzed hydrolysis of the dimetrhy ester of meso-cis-1,2-cyclohexanedicarboxylic acid yielded the optically pure (1S,2R)-monoester. The corresponding diethyl ester yielded racemic monoester.The diethyl ester of racemic trans-1,2-cyclohexanedicarboxylic acid was kinetically resolved by partial hydrolysis with subtilisin (EC 3.4.21.14) or pig liver esterase. The (1R,2R)-monoester had an enantiomeric excess of 45% and was obtained in an enantiomerically pure form through recrystallisation. The remaining (1S,2S)-diester exhibited an enantiomeric excess of 83%. The nature of the ester function (methyl, ethyl, and propyl esters) had a great influence on the enantiomeric excess obtained and on the kinetic parameters.     

A Novel Reductase from Candida albicans for the Production of Ethyl (S)-4-Chloro-3-hydroxybutanoate

A novel NADPH-dependent reductase (CaCR) from Candida albicans was cloned for the first time. It catalyzed asymmetric reduction to produce ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE). It contained an open reading frame of 843 bp encoding 281 amino acids. When co-expressed with a glucose dehydrogenase in Escherichia coli, recombinant CaCR exhibited an activity of 5.7 U/mg with ethyl 4-chloro-3-oxobutanoate (COBE) as substrate. In the biocatalysis of COBE to (S)-CHBE, 1320 mM (S)-CHBE was obtained without extra NADP⁺/NADPH in a water/butyl acetate system, and the optical purity of the (S)-isomer was higher than 99% enantiomeric excess.