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An enzymatic alternative to the chemical synthesis of chiral gem-difluorinated alcohols has been developed. The method is highly effective and stereoselective, feasible at laboratory temperature, avoiding the use of toxic heavy metal catalysts which is an important benefit in medicinal chemistry including the synthesis of drugs and drug precursors. Candida antarctica lipases A and B were applied for the enantioselective resolution of side-chain modified gem-difluorinated alcohols, (R)- and (S)-3-benzyloxy-1,1-difluoropropan-2-ols (1a and 1b), compounds serving as chiral building blocks in the synthesis of various bioactive molecules bearing a gem-difluorinated grouping. The catalytic activity of these lipases was investigated for the chiral acetylation of 1a and 1b in non-polar solvents using vinyl acetate as an acetyl donor. The dependence of the reaction course on various substrate and enzyme concentrations, reaction time, and temperature was monitored by chiral capillary electrophoresis (CE) using sulfobutyl ether ß-cyclodextrin as a stereoselective additive of the aqueous background electrolyte. The application of CE, NMR, and MS methods has proved that the complex enzyme effect of Candida antarctica lipase B leads to the thermodynamically stable (S)-enantiomer 1b instead of the expected acetylated derivatives. In contrast, the enantioselective acetylation of racemic alcohol 1 was observed as a kinetically controlled process, where (R)-enantiomer 1a was formed as the main product. This process was followed by enzymatic hydrolysis and chiral isomerisation. Finally, single pure enantiomers 1a and 1b were isolated and their absolute configurations were assigned from NMR analysis after esterification with Mosher's acids.

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The present study was conducted to purify lipase from indigenous Bacillus subtilis strain Kakrayal_1 (BSK-L) for enantioselective resolution of racemic-ketoprofen. The production of lipase (BSK-L) was optimized using Plackett-Burman and central composite design of response surface methodology (RSM). The optimized media containing olive oil (3.5%), MnSO4 (8 mM), CaCl2 (5 mM), peptone (20 g/l), pH (8), agitation (180 rpm) and temperature (37 °C) resulted in maximum lipase production of 7500 U/g of cell biomass. The lipase was purified using sequential method to an overall purification fold of 13% with 20% recovery, 882 U/mg specific activity and a molecular weight of 45 kDa. Optimal pH and temperature of purified lipase were found to be 8 and 37 °C, respectively. Furthermore, BSK-L displayed good stability with various organic solvents, surfactants and metal ions. K m and V max values of lipase were observed to be 2.2 mM and 6.67  mmoles of product formed/min/mg, respectively. The racemic ketoprofen butyl ester was hydrolyzed using lipase with 49% conversion efficiency and 69% enantiomeric excess (ee) which was superior to the commercially procured lipase (Candida antarctica lipase). Thus, this enzyme could be considered as a promising candidate for the pharmaceutical industry.

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This work studied the enantioselective resolution of 2-arylpropionic acid derivatives employing immobilization of lipase produced by Bacillus subtilis strain Kakrayal_1 (BSK-L). The efficient immobilization of lipase on modified silica gel was confirmed by Fourier transform infrared spectroscopy. Tethering of lipase facilitated the enhancement of physiochemical properties and stability of enzyme. Covalently immobilized enzyme retained 85% of residual activity even on reuse after 10th reaction cycle. Validation of immobilized lipase for enantioselective resolution of 2-arylpropionic acid derivatives led to 47.8% conversion efficiency with 87% enantiomeric excess (ee) for ketoprofen, and 27.3% conversion efficiency with 75% ee for flurbiprofen. The enantioselective resolution using immobilized lipase (BSK-L) was superior to free and commercially procured lipase, which suggest a potential application of immobilized lipase in the pharmaceutical/chemical industry.

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Since the amine groups are highly reactive toward various functional moieties, the formation of covalent bonds between a biomolecule and an insoluble amine-functionalized support is the most frequently used technique in immobilization protocols. A new approach based on the Ugi four-component reaction was used for immobilization of Rhizomucor miehei lipase (RML) as a model enzyme on amine-functionalized silica and silica nanoparticles (SBA-15). For this, the amine-modified supports were prepared and the structural properties of the functionalized supports, prior to and after functionalization were characterized by using IR, SEM, TGA, DTA, TEM. Immobilization of RML on the aminated carriers was performed under extremely mild conditions (25 °C, pH 7). The results revealed very rapid immobilization of 150 and 200 mg of RML on 1 g of silica-NH2 and SBA-NH2, respectively, producing 95-100% of immobilization yield. The specific activity and optimum pH of the immobilized preparations and the effect of temperature and co-solvents on their stabilities as well as the reusability of the derivatives were tested. The immobilized preparations were also used as enantioselective catalyst in kinetic resolution of racemic ibuprofen. Among them, Silica-RML showed the best selectivity with 92.2% enantiomeric excess (ee) and E-value of 33.9.

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Several banned substances are illegally used by athletes in racemic mixtures for performance enhancement. These include clenbuterol, methyl hexaneamine, methamphetamines, and amphetamines. Clenbuterol is present in a large number of doping samples from Olympic and non-Olympic athletes that have adverse analytical findings (AAFs). In some cases, the presence of these substances could be the result of consumption of meat contaminated with clenbuterol. In other cases, the origin is not clear. In this study, 27 products with racemic clenbuterol were evaluated using a new analytical methodology for the resolution of R-(-) and S-(+)-enantiomers of clenbuterol by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a chiral column in 15 min with good separation. The method developed can also be used for the analysis of other biological matrices such as urine, serum, and meat. The resolution between two peaks' (Rs ) value obtained using chromatographic data was 1.03. Both clenbuterol enantiomers were present in all products analyzed and the ratio was nearly 1. The origin of the product was not important for determining the presence of one or both enantiomers. All products displayed a 50:50 ratio of clenbuterol enantiomers. To the best of our knowledge, clenbuterol ratio determination of a large number of pharmaceutical preparations and black-market products has not been reported previously. The information shown could be used by national anti-doping organizations and the athletes with AAFs attributed to clenbuterol. Copyright © 2017 John Wiley & Sons, Ltd.

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Enantiomerically pure 3,3,3-trifluoro-2-hydroxy-2-methylpropionic acids are important chiral building blocks for a series of pharmaceuticals. Here, a bacteria strain with 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide-degrading ability was screened and identified as Burkholderia phytofirmans ZJB-15079, from which a novel amidase (Bp-Ami) was cloned and demonstrated to be capable of kinetic resolution of rac-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide to optically pure (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid. Phylogenetic analysis revealed that Bp-Ami was closely located to the acetamidase/formamidase (FmdA_AmdA) family, and it shared high homology with acetamidases. Bp-Ami was found to be the first cobalt-dependent FmdA_AmdA family amidase. The enzyme activity was significantly increased by 37.7-fold in the presence of 1 mM Co2+, with a specific activity of 753.5 U/mg, K m value of 24.73 mM, and k cat /K m value of 22.47 mM-1 s-1. As an enzyme from mesophile, Bp-Ami exhibited extreme thermostability with a half-life of 47.93 h at 80 °C, which was even superior to other reported amidases from thermophiles. The whole cell catalysis of 200 g/L 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide by Escherichia coli harboring Bp-Ami (5 g/L) resulted in 44 % yield and an enantiomeric excess (ee p) of 95 % within 10 min (E = 86). The high substrate tolerance, high specific activity, and extreme thermostability demonstrated the great potential of Bp-Ami for efficient biocatalytic synthesis of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid.

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The enantioselective resolution of (R,S)-2,3-dichloro-1-propanol ((R,S)-DCP) to (S)-DCP by whole cells of a recombinant Escherichia coli expressing halohydrin dehalogenase (HHDH) activity was limited by product inhibition. To solve this problem to improve the productivity of (S)-DCP, an n-heptane-aqueous biphasic system was adopted in this work. The influential operational parameters including phase volumetric ratio, buffer pH and reaction temperature were optimized. Under the optimal reaction conditions, significant improvements of substrate concentration and biocatalyst productivity (375 mM and 7.64 mmol (S)-DCP g(-1) cell) were achieved in this n-heptane-aqueous biphasic system compared with aqueous single-phase system (150 mM and 2.97 mmol g(-1)cell). The scale-up biosynthesis of (S)-DCP was successfully performed in a 2-L stirred reactor, resulting in a 128.8 mM (S)-DCP with enantiomeric excess of 99.1% and average productivity of 2.07 g (S)-DCPL(-1) h(-1), respectively.

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