Scale-up of the enantioselective reaction for the enzymatic resolution of (R,S)-ibuprofen

The reaction for the resolution of (R,S)-ibuprofen was scaled-up to yield gram quantities of (S)-ibuprofen. This was accomplished through two enantioselective reactions each catalysed by Novozym 435. In the first reaction, starting from 300 g of racemic ibuprofen, 88.9 g of enantioenriched (S)-ibuprofen with an enantiomeric excess of the order of 85% were produced. In the subsequent reaction, 75 g of the 85 % e.e. material were utilized to produce 38.4 g of (S)-ibuprofen with an enantiomeric excess of 97.5 %.     

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%.     

Enzymatic enantioselective transcyanation of silicon-containing aliphatic ketone with (S)-hydroxynitrile lyase from Manihot esculenta

(S)-Hydroxynitrile lyase from Manihot esculenta (MeHNL) was shown for the first time to be able to catalyze the enantioselective transcyanation of acetyltrimethylsilane (ATMS) with acetone cyanohydrin to form (S)-2-trimethylsilyl-2-hydroxyl-propionitrile in an aqueous/organic biphasic system. To better understand the reaction, various influential variables were examined. The most suitable organic phase, optimal buffer pH, aqueous phase content, shaking rate, temperature, concentration of ATMS, acetone cyanohydrin and crude enzyme were diisopropyl ether (DIPE), 5.4, 13% (v/v), 190 rpm, 40°C, 10 mM, 20 mM, and 35 U/ml, respectively, under which the initial reaction rate, substrate conversion and product enantiomeric excess (e.e.) were 19.5 mM/h, 99.0% and 93.5%, respectively. A comparative study demonstrated that silicon atoms in the substrate had a great effect on the reaction, and that ATMS was a much better substrate for MeHNL than its carbon analogue 3,3-dimethyl-2-butanone (DMBO) with respect to the initial reaction rate, substrate conversion and product e.e. MeHNL has greater affinity towards ATMS than its carbon analogue as indicated by the much lower K_m. The activation energy of MeHNL-catalyzed transcyanation of ATMS was also markedly lower than that of DMBO. The silicon effect on the reaction was rationalized on the basis of the special characteristics of silicon atoms and the catalytic mechanism of MeHNL.     

Parameters influencing asymmetric synthesis of (R)-mandelonitrile by a novel (R)-hydroxynitrile lyase from Eriobotrya japonica

(R)-Mandelonitrile was successfully synthesized by an enzymatic transcyanation reaction of benzaldehyde and acetone cyanohydrin catalyzed by a hydroxynitrile lyase from Eriobotrya japonica (EjHNL) in an aqueous-organic biphasic system. The effects of pH, temperature, organic solvent, substrate concentration and enzyme concentration on the initial activity and enantioselectivity of the enzyme were studied. Both pH and temperature had a large effect on the initial velocity and enantiomeric excess (e.e.) of the product, (R)-mandelonitrile. High enantiomeric purity of the product was observed at low pH and temperature because the non-enzymatic reaction producing racemates of mandelonitrile was almost suppressed. The optimum pH and temperature to obtain high e.e. were pH 4.0 and 10 °C, respectively. Surprisingly, the organic solvents had a significant influence on the initial velocity of the reaction but less influence on the enantiomeric purity of product. The EjHNL was very stable in ethyl acetate, diethyl ether, methyl-t-butyl ether, diisopropyl ether, dibutyl ether and hexane for 12 h. The best solvent for the highest initial velocity and e.e. was diethyl ether with an optimum aqueous phase content of 50% (v/v). The initial reaction rate increase as the aqueous phase content rose, but when the content was more than 50%, a reduction of e.e. was observed. Increasing the concentration of the substrates accelerated the initial velocity, but caused a slight decrease in the e.e. of the product. Under the optimized conditions, the conversion and e.e. of (R)-mandelonitrile for 3 h were 40 and 99%, respectively. The aqueous phase containing the enzyme also showed considerably efficient reusability for 4 batch reactions.     

Synthesis of (R)-2-trimethylsilyl-2-hydroxyl-propionitrile catalyzed by (R)-hydroxynitrile lyase from Prunus japonica seed meal

Synthesis of (R)-2-trimethylsilyl-2-hydroxyl-propionitrile via asymmetric transcyanation of acetyltrimethylsilane with acetone cyanohydrin in an aqueous/organic biphasic system catalyzed by (R)-hydroxynitrile lyase from Prunus japonica seed meal was successfully carried out for the first time. The optimal volume ratio of aqueous to organic phase, buffer pH value and reaction temperature were 15% (v/v), 5.0 and 30°C, respectively, under which both substrate conversion and product enantiomeric excess (ee) were 99%. Silicon atom in the substrate showed great effect on the reaction. Acetyltrimethylsilane was a much better substrate for (R)-hydroxynitrile lyase from Prunus japonica than its carbon analogue.     

A convenient method for the stereoselective preparation of 3-hydroxy-2-substituted propionaldehyde derivatives, C-terminal precursors for the synthesis of trans-alkene dipeptide isosteres

Summary (S)-3-hydroxy-2-substituted propionaldehyde dimethyl or diethyl acetals 3, which are versatile synthons in dipeptide isostere synthesis, were synthesized in 54–95% enantiomeric excess by reduction of (S,R)-acetalized acyloxazolidinones 7 with LiAlH₄.     

Asymmetric reduction of 3-chloropropiophenone to (S)-3-chloro-1-phenylpropanol using immobilized Saccharomyces cerevisiae CGMCC 2266 cells

(S)-3-Chloro-1-phenylpropanol is an important chiral precursor for numerous antidepressants such as tomoxetine. A high enantiomeric excess (e.e.) of (S)-3-chloro-1-phenylpropanol can be achieved by asymmetric reduction of 3-chloropropiophenone using Saccharomyces cerevisiae CGMCC 2266 cells immobilized in calcium alginate. Thermal pretreatment of the immobilized cells at 50 °C for 30 min resulted in high enantioselectivity (99% e.e.) and good percent conversion (80%). The effects of various conditions on the reduction reaction were investigated. The optimal conditions were found to be as follows: sodium alginate concentration, 2%; bead diameter, 2 mm; temperature, 30 °C; re-culture time, 24 h; and batch addition of the substrate. After reusing these three times, the immobilized cells retained approximately 60% of their original catalytic activity with their enantioselectivity intact.     

Screening for Antioxidants of Microbial Origin

Asymmetric hydrolysis of (dl)-1-acyloxy-2-halo-1-phenylethanes by lipoprotein lipase Amano P from Pseudomonas fluorescens and the lipase from Chromobacterium viscosum afforded the optically active (R) residual substrates and (S)-2-halo-1-hydroxy-1-phenylethanes in 100% enantiomeric excess (e.e.). The length of acyl residues from acetyl to octanoyl in the substrates did not influence the enantioselectivity.

Both enantiomers of optically active styrene oxides were synthesized from the enzymatic products.     

Enhanced Asymmetric Reduction of Ethyl 3‐Oxobutyrate by Baker's Yeast via Substrate Feeding and Enzyme Inhibition

The moderate enantioselectivity of wild form baker's yeast can be considerably increased either by using continuous feeding to maintain a low substrate concentration throughout the reaction, or by the selective inhibition of competing enzymatic pathways. The reduction of ethyl 3‐oxobutyrate to ethyl (S)‐3‐hydroxybutyrate was used as a model reaction. With the substrate feeding method, the enantioselectivity could be increased from 75 % to as high as 98 %. The increased selectivity originates from the much higher substrate binding constant of the (R)‐specific enzymes, so that these enzymes remain essentially inactive if a low concentration of ethyl 3‐oxobutyrate is maintained in the bioreactor. Alternatively, the enantioselectivity of baker's yeast can be improved by selectively blocking competing enzymatic pathways. It was found that vinyl acetate is a selective inhibitor for the (R)‐specific enzymes. Ethyl (S)‐3‐hydroxybutyrate with an enantiomeric excess of 98 % was obtained by pre‐incubation of baker's yeast in 100 mM of vinyl acetate solution for 1 h. These results suggest that by selecting appropriate process conditions, natural baker's yeast can be a competitive biocatalyst for the large‐scale production of chiral secondary alcohols.     

Improved enantioselectivity of E. coli BioH in kinetic resolution of methyl (S)-3-cyclohexene-1-carboxylate by combinatorial modulation of steric and aromatic interactions

As a chiral precursor for the important anticoagulant Edoxaban, enantioselective synthesis of (S)-3-cyclohexene-1-carboxylic acid is of great significance. The complicated procedures and generation of massive solid waste discourage its chemical synthesis, and the alternative biocatalysis route calls for an enzyme capable of asymmetric hydrolysis of racemic methyl-3-cyclohexene-1-carboxylate. To this end, we engineered the E. coli esterase BioH for improved S-enantioselectivity via rational design. By combinatorial modulation of steric and aromatic interactions, a positive mutant Mu3 (L24A/W81A/L209A) with relatively high S-selectivity in hydrolyzing racemic methyl-3-cyclohexene-1-carboxylate was obtained, improving the enantiomeric excess from 32.3% (the wild type) to 70.9%. Molecular dynamics simulation was conducted for both (R)- or (S)- complexes of the wild type and Mu3 to provide hints for the mechanism behind the increased S-selectivity. Moreover, the reaction conditions of Mu3 in methyl-3-cyclohexene-1-carboxylate hydrolysis was optimized to improve the conversion rate to 2 folds.     

A novel lipase from Aspergillus oryzae catalyzed resolution of (R,S)-ethyl 2-bromoisovalerate

In this study, a novel lipase M5 derived from Aspergillus oryzae WZ007 was prone to exhibit high hydrolytic activity and stereoselectivity towards racemic substrate (R,S)-ethyl 2-bromoisovalerate. (R)-ethyl 2-bromoisovalerate was obtained by enzymatic resolution, which is the key chiral intermediate for highly efficient enantiomerically fluvalinate. The results showed that the enzymatic reaction was carried out in 120mM racemic substrate for 3 hours, the enantiomeric excess reached 98.6%, the conversion was 51.7%, and E value above 120. Therefore, the novel lipase M5 has the ability to efficiently produce (R)-ethyl 2-bromoisovalerate, which greatly reduces the industrial production cost of the highly efficient counterpart of fluvalinate.     

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.     

Enzymatic resolution of homoallyllic alcohols using various Rhizopus species

The asymmetric resolution of various 1-aryl-3-buten-1-ols via microbial hydrolysis of the corresponding acetates has been investigated using different Rhizopus species. The chosen species, R. arrhizus (wild type), efficiently hydrolyzed 1-phenyl- and 1-para-substituted phenyl-3-buten-1-ol acetates, producing the enantiomerically pure (R)-alcohols with 53–65% yields. Although the antipode acetates were obtained with 9–52% enantiomeric excess, the (S)-alcohols were amenable in > 99% enantiomeric excess via a R. arrhizus mediated asymmetric reduction of the corresponding ketones.     

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.     

Exploration of the expeditious potential of Pseudomonas fluorescens lipase in the kinetic resolution of racemic intermediates and its validation through molecular docking

A profoundly time‐efficient chemoenzymatic method for the synthesis of (S)‐3‐(4‐chlorophenoxy)propan‐1,2‐diol and (S)‐1‐chloro‐3‐(2,5‐dichlorophenoxy)propan‐2‐ol, two important pharmaceutical intermediates, was successfully developed using Pseudomonas fluorescens lipase (PFL). Kinetic resolution was successfully achieved using vinyl acetate as acylating agent, toluene/hexane as solvent, and reaction temperature of 30°C giving high enantioselectivity and conversion. Under optimized condition, PFL demonstrated 50.2% conversion, enantiomeric excess of 95.0%, enantioselectivity (E = 153) in an optimum time of 1 hour and 50.3% conversion, enantiomeric excess of 95.2%, enantioselectivity (E = 161) in an optimum time of 3 hours, for the two racemic alcohols, respectively. Docking of the R‐ and S‐enantiomers of the intermediates demonstrated stronger H‐bond interaction between the hydroxyl group of the R‐enantiomer and the key binding residues of the catalytic site of the lipase, while the S‐enantiomer demonstrated lesser interaction. Thus, docking study complemented the experimental outcome that PFL preferentially acylated the R form of the intermediates. The present study demonstrates a cost‐effective and expeditious biocatalytic process that can be applied in the enantiopure synthesis of pharmaceutical intermediates and drugs.     

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.     

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 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.     

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.     

Optimization of (R,S)-1-phenylethanol kinetic resolution over Candida antarctica lipase B in ionic liquids

The kinetic resolution of racemates constitutes one major route to manufacture optically pure compounds. The enzymatic kinetic resolution of (R,S)-1-phenylethanol over Candida antarctica lipase B (CALB) by using vinyl acetate as the acyl donor in the acylation reaction was chosen as model reaction. A systematic screening and optimization of the reaction parameters, such as enzyme, ionic liquid and substrates concentrations with respect to the final product concentration, were performed. The enantioselectivity of immobilized CALB commercial preparation, Novozym 435, was assayed in several ionic liquids as reaction media. In particular, three different ionic liquids: (i) 1-butyl-3-methylimidazolium hexafluorophosphate [bmim][PF₆], (ii) 1-butyl-3-methylimidazolium tetrafluoroborate [bmim][BF₄] and (iii) 1-ethyl-3-methylimidazolium triflimide [emim][NTf₂] were tested. At 6.6% (w/w) of Novozym 435, dispersed in 9.520 M of [bmim][PF₆] at 313.15 K, using an equimolar ratio of vinyl acetate/(R,S)-1-phenylethanol after 3 h of bioconversion, the highest possible conversion (50%) was reached with enantiomeric excess for substrate higher than 99%.