- Efficient access to l-phenylglycine using a newly identified amino acid dehydrogenase from: Bacillus clausii
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An amino acid dehydrogenase from Bacillus clausii (BcAADH) was identified and overexpressed in Escherichia coli BL21(DE3) for the preparation of l-phenylglycine from benzoylformic acid. Recombinant BcAADH was purified to homogeneity and characterized. BcAADH could catalyse reductive amination and oxidative deamination at optimum pHs of 9.5 and 10.5. Furthermore, BcAADH has a broad substrate spectrum, displaying activities toward various aromatic and aliphatic keto acids. When coexpressed with glucose dehydrogenase from Bacillus megaterium, the potential application of BcAADH in the preparation of l-phenylglycine was investigated at a high substrate loading and low biocatalyst addition. As much as 400 mM benzoylformic acid could be fully reduced into l-phenylglycine within 6 h at >99.9% ee. With merely 0.5 g DCW L-1, 200 mM benzoylformic acid was completely reduced, resulting in a substrate to biocatalyst ratio of 60 g g-1, environmental factor of 4.7 and 91.7% isolation yield at gram scale. This study provides guidance for the application of BcAADH in the synthesis of chiral non-natural amino acids.
- Cheng, Jun,Xu, Guochao,Han, Ruizhi,Dong, Jinjun,Ni, Ye
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- Resolution of racemic 2-chlorophenyl glycine with immobilized penicillin G acylase
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Racemic 2-chlorophenyl glycine has been resolved to obtain (S)-α-amino-(2-chlorophenyl)acetic acid with >99% enantiomeric purity via enantioselective hydrolysis of its N-phenylacetyl derivative with penicillin G acylase immobilized on Eupergit C. The reso
- Fadnavis, Nitin W.,Vedamayee Devi, Avala,Swarnalatha Jasti, Lakshmi
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- Stereoselective synthesis of l-tert-leucine by a newly cloned leucine dehydrogenase from Exiguobacterium sibiricum
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A leucine dehydrogenase from Exiguobacterium sibiricum (EsLeuDH) was discovered by genome mining approach. The EsLeuDH was overexpressed in Escherichia coli BL21, purified to homogeneity and characterized. This enzyme showed good thermostability with a half-life of 3.1 h at 60 °C. Furthermore, EsLeuDH has a broad spectrum of substrate specificity, showing activities toward many aliphatic α-keto acids and L-amino acids, in addition to some aryl α-keto acids and aryl α-amino acids, such as α-oxobenzeneacetic and l-phenylglycine. The EsLeuDH was successfully coexpressed with Bacillus megaterium glucose dehydrogenase (BmGDH) in Escherichia coli BL21 for the production of l-tert-leucine. By using the coexpressed whole cells, a decagram preparation of l-tert-leucine was performed at a substrate concentration of 0.6 M (78.1 g L-1) in 1 L scale with 99% conversion after 5.5 h, resulting in 80.1% yield and > 99% ee (enantiomeric excess).2014 Published by Elsevier B.V.
- Li, Jing,Pan, Jiang,Zhang, Jie,Xu, Jian-He
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- Method for continuously and quickly preparing DL-phenylglycine and analogue thereof
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The invention provides a method for continuously and quickly preparing DL-phenylglycine and an analogue thereof. The method comprises the steps of adding 2-hydroxyl-phenylacetonitrile and an analoguethereof (cyanohydrin for short) and an aqueous ammonium bicarbonate solution into a microchannel reactor for a reaction, controlling the reaction temperature to be 80-130 DEG C, and controlling the reaction pressure to be 0.5-2.0 MPa, wherein the standing time of the reactants in a microchannel is 1-8 min, and an aqueous solution of 5-phenyl-hydantoin and an analogue thereof (hydantoin for short)is obtained; adding the hydantoin and alkali into the microchannel reactor for a reaction, controlling the reaction temperature to be 120-200 DEG C, and controlling the reaction pressure to be 1.0-3.5MPa, wherein the standing time of the reactants in the microchannel is 1-8 min, and then a saline solution of phenylglycine and an analogue thereof is obtained; conducting acidification neutralization and crystallization to obtain the phenylglycine and the analogue thereof. According to the method, the microchannel reactor is adopted, the reaction time is greatly shorted, the reaction speed is increased, pyrolysis and polymerization of the cyanohydrin are reduced, no by-products are generated, the products are high in yield, clean and environmentally friendly, and the production cost is lowered.
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Paragraph 0031-0033
(2019/07/04)
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- Preparation method for D, L-phenylglycine and analogue thereof
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The invention provides a preparation method for D, L-phenylglycine and an analogue thereof. According to the method, benzaldehyde, an analogue thereof and hydrocyanic acid are adopted as raw materials and subjected to cyanidation reaction, and then 2-hydroxy-benzyl cyanide or 2-hydroxy-benzyl cyanide analogue (cyanohydrin for short) is generated. Cyanohydrin reacts with carbon dioxide and the aqueous solution of ammonia, and then 5-phenyl-hydantoin and an analogue thereof (hydantoin for short) are generated. hydantoin is successively subjected to steam stripping, alkaline hydrolysis, steam stripping, decolorization, neutralization, crystallization, washing, centrifuging, drying and the like to obtain D, L-phenylglycine and the analogue thereof. Compared with the prior art, the preparation method for D, L-phenylglycine and the analogue thereof can significantly and effectively reduce the pollution, and fewer inorganic salt by-products are generated. Meanwhile, the prepared D, L-phenylglycine and the analogue thereof are high in product yield and high in purity. Counted in benzaldehyde and the analogue thereof, the yield of D, L-phenylglycine and the analogue thereof is larger than or equal to 96%, and the product purity is larger than or equal to 99%. Meanwhile, the process flow is simple and feasible, so that the method is worthy of market popularization and application.
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Paragraph 0049; 0050
(2017/03/17)
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- Chemo-enzymatic approach to the synthesis of the antithrombotic clopidogrel
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The (S)-2-chlorophenylglycine moiety is well recognized in the structure of (S)-clopidogrel, a known antithrombotic drug. We prepared an enantiomerically pure chiral building block via an enzyme-catalyzed resolution of (RS)-N-Boc-2-chlorophenylglycine methylester. The best results were obtained by means of an immobilized subtilisin, the cross-linked enzyme aggregate (Alcalase-CLEA). The high enantiomeric excess of the synthon obtained remained the same over the course of clopidogrel synthesis; the simplicity of the process makes this pathway suitable for large-scale preparation.
- Ferraboschi, Patrizia,Mieri, Maria De,Galimberti, Fiorella
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experimental part
p. 2136 - 2141
(2010/10/03)
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- Process for preparation of 2-chlorophenylglycine derivatives and enantiomerically separation
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The present invention relates to novel processes and intermediates for the preparation of R(?)-α-2-chlorophenylglycine, S(+)-α-2-chlorophenylglycine, and RS-2-chlorophenylglycine derivatives of formulas I, II and III, respectively. The resulting enantioseparative system was validated in order to evaluate the presence of the enantiomer in pharmaceutical samples. These compounds are found useful as an active ingredient for the pharmaceutical intermediate or as an active ingredient as the tools for delivery of drugs.
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