Enantioselective esterification of ibuprofen in supercritical carbon dioxide by immobilized lipase

The enantioselective esterification of racemic ibuprofen with n-propanol by immobilized Mucor miehel lipase in supercritical carbon dioxide was studied. The enantiomeric excess of the product (ee_p) was 70 % at 15...20 % conversion. The enantioselectivity was faintly affected by temperature and the concentration of ibuprofen and lipase. The optimum temperature was 45 °C. The initial reaction rate increased with pressure, but enantioselectivity was not affected by pressure changes. The reaction rates in supercritical carbon dioxide at optimized conditions and in n-hexane were similar.     

Experiment and simulation on kinetic resolution of (R,S)-2-chloromandelic acid by enzymatic transesterification

Optically pure 2-chloromandelic acid (ClMA) is a very important chiral drug intermediate for synthesis of (S)-clopidogrel, belonging to the platelet aggregation inhibitor. Enantioselective resolution of (R,S)-2-chloromandelic acid was carried out in organic solvent through irreversible transesterification catalyzed by lipase AK with vinyl acetate acting as the acyl donor. Effects of various conditions on enantioselectivity and activity of lipase were investigated, including organic solvents, temperature, water content, substrate ratio, enzyme loading, and reaction time. Based on homogeneous reaction and Ping-Pong bi-bi mechanism, a quantitative model was constructed to simulate and optimize the reaction process. Under the optimal conditions, excellent results were obtained with high conversion of (R)-2-ClMA (c _R, ≥98.85%) and large enantiomeric excess of substrate (ee _s, ≥98.15%). There is a good agreement between predicted values and experiment data, which indicates that the established method is a powerful tool for optimization of the enantioselective transesterification process for enantiomers separation.     

Lipase-catalyzed naproxen methyl ester hydrolysis in water-saturated ionic liquid: significantly enhanced enantioselectivity and stability

The lipase selective hydrolysis of Naproxen methyl ester was explored in both water-saturated isooctane and water-saturated ionic liquid 1-butyl-3-methylimidazolium hexafluoro-phoshate ([bmim]PF₆) to see any significant differences in terms of enantioselectivity and stability between two different classes of reaction media. It is shown that polar and hydrophobic of [bmim]PF₆ made it an unearthly reaction medium for hydrolysis of Naproxen methyl ester. It not only decreases the equilibrium constant (K) and enhances the enantiomeric ratio (E), consequently improves the equilibrium conversion (C_Eq) of the hydrolysis reaction and enantiomeric excess of product (ee_p), but also maintains the lipase activity. Because the lipase would not dissolve in the 1-butyl-3-methylimidazolium hexafluoro-phoshate, it can be filtrated up from 1-butyl-3-methylimidazolium hexafluoro-phoshate and recycled for several runs. The stability of lipase was improved due to the higher solubility of methanol in 1-butyl-3-methylimidazolium hexafluoro-phoshate than in isooctane.     

Simultaneous monitoring of the enantiomeric purities of both substrate and product in the enzymatic resolution of (R,S)-2-acetoxy-3-bromopropyl para-toluenesulfonate by HPLC chiral column chromatography

The enantiomeric purities of both substrate, 2-acetoxy-3-bromopropyl para-toluenesulfonate (I), and product, 2-hydroxy-3-bromopropyl p-toluenesulfonate (II) were examined in one analysis. The enzymatic resolution was conducted by Amano lipase AK and the enantiomeric excess as well as the conversion rate were monitored by HPLC analysis utilizing a Chiralcel OD column. After 7 h of reaction, HPLC results indicated that the enantiomeric purities of both substrate (I) and product (II) were greater than 95% and the conversion rate was around 55%. © 1995 Wiley-Liss, Inc.     

Copper‐Chiral Camphor β‐Amino Alcohol Complex Catalyzed Asymmetric Henry Reaction

Four novel chiral amino alcohols were synthesized from D‐(+)‐camphor and utilized as ligands in a Cu(I)‐catalyzed asymmetric Henry reaction. The reactions were carried out under mild conditions with excellent enantioselectivities and moderate yields without the exclusion of air or moisture. The highest enantioselectivity was observed up to 94% enantiomeric excess (ee) with ligand L1 in toluene at room temperature. Chirality 27:761–765, 2015. © 2015 Wiley Periodicals, Inc.     

Resolution of 2-nitroalcohols by Burkholderia cepacia lipase-catalyzed enantioselective acylation

Racemic 2-nitro-1-phenylethanol was resolved by via enantioselective transesterification catalyzed by Burkholderia cepacia lipase. The reaction afforded excellent E values (E > 200) and enantioselectivity (up to >99% enantiomeric excesses [ee]) of both remaining substrates and acetylated product. Moreover, the lipase displayed high enantioselectivity in the resolution of additional 2-nitroalcohols (E up to >200). This method provides an efficient alternative for obtaining enantiopure 2-nitroalcohols.     

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.     

Lipase‐Catalyzed Kinetic Resolution of (±)‐1‐(2‐Furyl) Ethanol in Nonaqueous Media

S‐1‐(2‐Furyl) ethanol serves as an important chiral building block for the preparation of various natural products, fine chemicals, and is widely used in the chemical and pharmaceutical industries. In this work, lipase‐catalyzed kinetic resolution of (R/S)‐1‐(2‐furyl) ethanol using different acyl donors was investigated. Vinyl esters are good acyl donors vis‐à‐vis alkyl esters for kinetic resolution. Among them, vinyl acetate was found to be the best acyl donor. Different immobilized lipases such as Rhizomucor miehei lipase, Thermomyces lanuginosus lipase, and Candida antarctica lipase B were evaluated for this reaction, among which C. antarctica lipase B, immobilized on acrylic resin (Novozym 435), was found to be the best catalyst in n‐heptane as solvent. The effect of various parameters was studied in a systematic manner. Maximum conversion of 47% and enantiomeric excess of the substrate (ee_s) of 89% were obtained in 2 h using 5 mg of enzyme loading with an equimolar ratio of alcohol to vinyl acetate at 60°C at a speed of 300 rpm in a batch reactor. From the analysis of progress curve and initial rate data, it was concluded that the reaction followed the ordered bi–bi mechanism with dead‐end ester inhibition. Kinetic parameters were obtained by using nonlinear regression. This process is more economical, green, and easily scalable than the chemical processes. Chirality 26:286–292, 2014. © 2014 Wiley Periodicals, Inc.     

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%.     

Biocatalytic synthesis of chiral N-(2-hydroxyalkyl)-acrylamides

Abstract

The preparation of a series of novel chiral N-(2-hydroxylalkyl)-acrylamides through a lipase-catalyzed resolution of racemic alkanolamines is described. The absolute stereochemistry and enantiomeric excess of the products were determined by a modified Mosher's method. The method was validated for this particular case by the synthesis of an enantiomerically pure product. Moreover, the stereoselective behavior of the lipase in this reaction is discussed.     

Enantioselective lipase-catalyzed ester hydrolysis: Effects on rates and enantioselectivity from a variation of the ester structure

In order to make a preliminary study of substituent effects on the rate and enantioselectivity obtained in esterolytic reactions catalyzed by a lipase from Candida rugosa, a series of racemic esters, derived from some α-alkyl and α-halo phenylacetic acids, were prepared. The reactions were studied at pH 6.0 and 50°C under which conditions uncatalyzed hydrolysis was relatively slow. Reaction samples were studied at different points of time by means of analytical chiral reversed-phase liquid chromatography, which permitted the simultaneous determination of product enantiomeric excess and of the degree of total ester hydrolysis. These data were then used to calculate initial rates as well as enantioselectivity. An increase of the steric bulk of the α-substituent was found to highly decrease the rate of the reaction. On the other hand, rates were higher for the p-nitrophenyl esters than for the corresponding 2-chloroethyl esters. Consistently, the enantioselectivity was found to be higher for the latter type of ester. The esters of the α-halo (bromo and chloro) phenylacetic acids gave mandelic acid as the final product. This was caused by a rapid solvolysis of the α-halo phenylacetic acid initially formed. © 1993 Wiley-Liss, Inc.     

Lipase-catalyzed Kinetic Resolution of (±)-trans- and cis-2-Azidocycloalkanols

The lipase-catalyzed kinetic resolution of trans- and cis-2-azidocycloalkanols and the preparation of enantiomerically pure trans- and cis-2-aminocycloalkanols are described.

Four kinds of lipases were screened for the acetylation of trans- and cis-2-azidocycloalkanols. Among them, Pseudomonas sp. lipases (lipase PS and lipase AK, Amamo Pharmaceutical Co.) showed the highest enantioselectivity. These products were converted to the corresponding 2-aminocycloalkanols to determine their enantiomeric excess (ee) and absolute configurations by HPLC and CD analyses, using (S)-TBMB carboxylic acid [(S)-2-tert-butyl-2-methyl-1,3-benzodioxole-4-carboxylic acid] as the chiral conversion reagent. The results of the CD analysis proved N,O-bis-(S)-TBMB carboxylated cis-2-aminocycloalkanols to adopt a predominantly N-equatorial conformation.

The partially resolved trans- and cis-2-aminocycloalkanols, except for trans-2-aminocyclopentanol, were recrystallized from ethyl acetate to give enantiomerically pure forms.     

ENANTIOSELECTIVE ACYLATION OF β-PHENYLALANINE ACID AND ITS DERIVATIVES CATALYZED BY PENICILLIN G ACYLASE FROM Alcaligenes faecalis

This study developed a simple, efficient method for producing racemic β-phenylalanine acid (BPA) and its derivatives via the enantioselective acylation catalyzed by the penicillin G acylase from Alcaligenes faecalis (Af-PGA). When the reaction was run at 25°C and pH 10 in an aqueous medium containing phenylacetamide and BPA in a molar ratio of 2:1, 8 U/mL enzyme and 0.1 M BPA, the maximum BPA conversion efficiency at 40 min only reached 36.1%, which, however, increased to 42.9% as the pH value and the molar ratio of phenylacetamide to BPA were elevated to 11 and 3:1, respectively. Under the relatively optimum reaction conditions, the maximum conversion efficiencies of BPA derivatives all reached about 50% in a relatively short reaction time (45–90 min). The enantiomeric excess value of product (ee_p ) and enantiomeric excess value of substrate (ee_s ) were all above 98% and 95%, respectively. These results suggest that the method established in this study is practical, effective, and environmentally benign and may be applied to industrial production of enantiomerically pure BPA and its derivatives.     

Improvement of the enantioselectivity and activity of lipase from Pseudomonas sp. via adsorption on a hydrophobic support: kinetic resolution of 2-octanol

Pseudomonas sp. lipase (PSL) was successfully immobilized on a novel hydrophobic polymer support through physical adsorption and the immobilized PSL was used for resolution of (R,S)-2-octanol with vinyl acetate as acyl donor. Enhanced activity and enantioselectivity were observed from the immobilized PSL compared with free PSL. The effects of reaction conditions such as temperature, water activity, substrate molar ratio and the amount of immobilized lipase were investigated. Under optimum conditions, the residual (S)-2-octanol was recovered with 99.5% enantiomeric excess at 52.9% conversion. The results also indicated that the immobilized PSL could maintain 94% of its initial activity even after reusing it five times.     

Improved production of (R)-2-octanol via asymmetric reduction of 2-octanone with Oenococcus oeni CECT4730 in a biphasic system

Abstract

Oenococcus oeni CECT4730, which catalyses the asymmetric reduction of 2-octanone to (R)-2-octanol with high enantioselectivity, was further studied to exploit its potential for production of (R)-2-octanol in an aqueous/organic solvent biphasic system. Variables such as the volume ratio of aqueous to organic phase (V_a/V_o), buffer pH, reaction temperature, shaking speed, co-substrates and the ratio of biocatalyst to substrate were examined with respect to the molar conversion, the initial reaction rate and the product enantiomeric excess (e.e.). Under the optimized conditions (V_a/V_o=1:1 (v/v), buffer pH=8.0, reaction temperature=30°C, shaking speed=150 rev/min, ratio of glucose to biomass=5.4:l (w/w), ratio of biocatalyst to substrate=0.51:l (g/mol)), the highest space time yield of (R)-2-octanol, 24 mmol L^?1 per h, and >98% product e.e. were obtained at a substrate concentration close to 1.0 mol L^?1 after 24 h reduction.     

Mandelic acid chiral separation utilizing a two-phase partitioning bioreactor built by polysulfone microspheres and immobilized enzymes

A novel two-phase partitioning bioreactor (TPPB) modified by polysulfone (PSF) microspheres and immobilized enzyme (novozym-435) was formed, and the resulting TPPB was applied into mandelic acid chiral separation. The PSF microspheres containing n-hexanol (named PSF/hexanol microspheres) was prepared by using the phase inversion method, which was used as the organic phase. Meanwhile, the immobilized enzyme novozym-435 was used as a biocatalyst. The water phase was composed of the phosphate buffer solution (PBS). (R, S)-Methyl mandelate was selected as the substrate to study enzymatic properties. Different reaction factors have been researched, such as pH, reaction time, temperature and the quantity of biocatalyst and PSF/hexanol microspheres added in. Finally, (S)-mandelic acid was obtained with an 80 % optical purity after 24 h in the two-phase partitioning bioreactor. The enantiomeric excess (ee_p) values were very low in the water phase, in which the highest ee_p value was only 46 %. The ee_p of the two-phase partitioning bioreactor had been enhanced more obviously than that catalyzed in the water phase.     

A simple method to determine concentration of enantiomers in enzyme-catalyzed kinetic resolution

Kinetic resolutions play important roles in industrial biotransformations for production of optical pure compounds from racemic substrates. A simple method, based on enantiomeric excess of both substrate (ee _S) and the corresponding product (ee _P), was developed for determination of concentration of enantiomers in kinetic resolution. Since only relative quantity (ee) was required in the proposed method, calibration and cumbersome quantitative sample handling can be avoided and analytical accuracy can be greatly improved.     

Immobilization of Candida rugosa lipase on glass beads for enantioselective hydrolysis of racemic Naproxen methyl ester

Candida rugosa lipase (CRL) was immobilized on glutaraldehyde-activated aminopropyl glass beads by using covalent binding method or sol-gel encapsulation procedure and improved considerably by fluoride-catalyzed hydrolysis of mixtures of RSi(OCH₃)₃ and Si(OCH₃)₄. The catalytic properties of the immobilized lipases were evaluated into model reactions, i.e. the hydrolysis of p-nitrophenylpalmitate (p-NPP). It has been observed that the percent activity yield of the encapsulated lipase was 166.9, which is 5.5 times higher than that of the covalently immobilized lipase. The enantioselective hydrolysis of racemic Naproxen methyl ester by immobilized lipase was studied in aqueous buffer solution/isooctane reaction system and it was noticed that particularly, the glass beads based encapsulated lipases had higher conversion and enantioselectivity compared to covalently immobilized lipase. In short, the study confirms an excellent enantioselectivity (E > 400) for the encapsulated lipase with an ee value of 98% for S-Naproxen.     

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.     

Enzymatic production of Cilastatin intermediate via highly enantioselective hydrolysis of methyl (±)-2,2-dimethylcyclopropane carboxylate using newly isolated Rhodococcus sp. ECU1013

(S)-(+)-2,2-Dimethylcyclopropane carboxylic acid [(S)-(+)-DMCPA] is a key chiral intermediate for production of Cilastatin, an excellent renal dehydropeptidase-I inhibitor. In this study, a new method for preparation of (S)-(+)-DMCPA with microbial esterases was investigated. A microbial screening program obtained six esterase-producing isolates that could display relatively high activities and enantioselectivities using racemic ethyl 2,2-dimethylcyclopropane carboxylate (DMCPE) as screening substrate, aiming at forming optically pure (S)-(+)-DMCPA. Further selection was carried out with substrates having different alcohol moieties, including methyl, ethyl, and 2-chloroethyl esters. Finally, one of these strains, numbered ECU1013, with high enantioselectivity toward the hydrolytic resolution of methyl 2,2-dimethylcyclopropane carboxylate (DMCPM), afforded the (S)-product in 92 % ee, and was later identified as Rhodococcus sp. According to our research, there were several active esterases to DMCPM in cells of Rhodococcus sp. ECU1013; however, (S)-preferential esterase was selectively enriched based on the time-dependent profile of esterases biosynthesis, thereby the enantiomeric excess of biotransformation product (ee _p) was constantly increased, finally maintained at 95 % (S). To improve the yield, various organic solvents were employed for better dispersion of the hydrophobic substrate. As a result, (±)-DMCPM of up to 400 mM in the organic phase of isooctane was enantioselectively hydrolyzed into (S)-(+)-DMCPA, with an isolation yield of 38 % and a further increase of ee _p to 99 %.