- Deracemization of unnatural amino acid: Homoalanine using d-amino acid oxidase and ω-transaminase
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A deracemization method was developed to generate optically pure l-homoalanine from racemic homoalanine using d-amino acid oxidase and ω-transaminase. A whole cell reaction using a biphasic system converted 500 mM racemic homoalanine to 485 mM l-homoalanine (>99% ee). The Royal Society of Chemistry 2012.
- Seo, Young-Man,Mathew, Sam,Bea, Han-Seop,Khang, Yong-Ho,Lee, Sang-Hyeup,Kim, Byung-Gee,Yun, Hyungdon
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- Synthesis of 4-hydroxyisoleucine by the aldolase-transaminase coupling reaction and basic characterization of the aldolase from Arthrobacter simplex AKU 626
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Arthrobacter simplex AKU 626 was found to synthesize 4-hydroxyisoleucine from acetaldehyde, α-ketobutyrate, and L-glutamate in the presence of Escherichia coli harboring the branched chain amino acid transaminase gene (ilvE) from E. coli K12 substrain MG-1655. By using resting cells of A. simplex AKU 626 and E. coli BL21(DE3)/pET-15b-ilvE, 3.2 mM 4-hydroxyisoleucine was produced from 250 mM acetaldehyde, 75 mM α-ketobutyrate, and 100mM L-glutamate with a molar yield to α-ketobutyrate of 4.3% in 50 mM Tris-HCl buffer (pH 7.5) containing 2 mM MnCl2·4H2O at 28°C for 2 h. An aldolase that catalyzes the aldol condensation of acetaldehyde and α-ketobutyrate was purified from A. simplex AKU 626. Mn2+ and pyridoxal 5′-monophosphate were effective in stabilizing the enzyme. The native and subunit molecular masses of the purified aldolase were about 180 and 32 kDa respectively. The N-terminal amino acid sequence of the purified enzyme showed no significant homology to known aldolases.
- Ogawa, Jun,Yamanaka, Hiroyuki,Mano, Junichi,Doi, Yuko,Horinouchi, Nobuyuki,Kodera, Tomohiro,Nio, Noriki,Smirnov, Sergey V.,Samsonova, Natalya N.,Kozlov, Yury I.,Shimizu, Sakayu
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- Nonproteinogenic α-amino acid preparation using equilibrium shifted transamination
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Microbial α-transaminases such as tyrosine aminotransferase (TAT) and branched chain aminotransferase (BCAT) of Escherichia coli, are useful as industrial biocatalysts to prepare nonproteinogenic L-amino acids from α-keto acids and an amino donor. However, they typically yield only 50% product when L-glutamic acid, the preferred amino donor, is used due to accumulated 2-ketoglutaric acid. Accordingly, methods have been sought to increase the reaction yield by the recycle or removal of the keto acid by-product. In this report, we have investigated the biocatalytic coupling of δ-transamination with α-transamination to recycle 2-ketoglutaric acid, and thereby increase the yield of aminotransferase reaction products. Ornithine δ-aminotransferase (OAT) catalyses the reversible transfer of the δ-amino group of L-ornithine to 2-ketoglutaric acid forming L-glutamic acid semialdehyde and L-glutamic acid. The cyclisation of L-glutamic acid semialdehyde to form Δ1-pyrroline-5-carboxylate under physiological conditions, favours the reaction in the direction of L-glutamic acid formation. The Bacillus subtilis rocD gene encoding OAT was cloned and produced at high levels in E. coli. Combined cell extracts of separate E. coli strains overproducing OAT and E. coli tyrosine aminotransferase enabled the synthesis of L-2-aminobutyrate from 2-ketobutyric acid to reach a yield of 92% compared to 50% achievable by TAT alone. Similarly, combined extracts of strains overproducing OAT and E. coli branched-chain amino acid aminotransferase synthesised L-tert-leucine from trimethylpyruvic acid with a 73% yield compared to 31% with BCAT alone. The use of OAT as a general biocatalytic tool to achieve high yields in aminotransferase reactions is discussed.
- Li, Tao,Kootstra, Anna B.,Fotheringham, Ian G.
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- Structure-guided engineering of: Pseudomonas dacunhae l-aspartate β-decarboxylase for l-homophenylalanine synthesis
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Structure-guided engineering of Pseudomonas dacunhael-aspartate β-decarboxylase (AspBDC) resulted in a double mutant (R37A/T382G) with remarkable 15400-fold improvement in specific activity reaching 216 mU mg-1, towards the target substrate 3(R)-benzyl-l-aspartate. A novel strategy for enzymatic synthesis of l-homophenylalanine was developed by using the variant as a biocatalyst affording 75% product yield within 12 h. Our results underscore the potential of engineered AspBDC for the biocatalytic synthesis of pharmaceutically relevant and value added unnatural l-amino acids.
- Zhang, Min,Hu, Pengfei,Zheng, Yu-Cong,Zeng, Bu-Bing,Chen, Qi,Zhang, Zhi-Jun,Xu, Jian-He
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- The specificity and kinetic mechanism of branched-chain amino acid aminotransferase from Escherichia coli studied with a new improved coupled assay procedure and the enzyme's potential for biocatalysis
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Branched-chain amino acid aminotransferase (BCAT) plays a key role in the biosynthesis of hydrophobic amino acids (such as leucine, isoleucine and valine), and its substrate spectrum has not been fully explored or exploited owing to the inescapable restrictions of previous assays, which were mainly based on following the formation/consumption of the specific branched-chain substrates rather than the common amino group donor/acceptor. In our study, detailed measurements were made using a novel coupled assay, employing (R)-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans as an auxiliary enzyme, to provide accurate and reliable kinetic constants. We show that Escherichia coli BCAT can be used for asymmetric synthesis of a range of non-natural amino acids such as l-norleucine, l-norvaline and l-neopentylglycine and compare the kinetic results with the results of molecular modelling. A full two-substrate steady-state kinetic study for several substrates yields results consistent with a bi-bi ping-pong mechanism, and detailed analysis of the kinetic constants indicates that, for good 2-oxoacid substrates, release of 2-oxoglutarate is much slower than release of the product amino acid during the transamination reaction. The latter is in fact rate-limiting under conditions of substrate saturation.
- Yu, Xuejing,Wang, Xingguo,Engel, Paul C.
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- Racemic Structures of Organic Ammonium Salts of N-Acetyl-DL-2-aminobutyric Acid and N-Acetyl-DL-norvaline and Optical Resolution by Preferential Crystallization of DL-Ammonium Salts
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The racemic structures of the ammonium salts (AM salts) and seven organic ammonium salts of N-acetyl-DL-2-aminobutyric acid (Dl-AcAbu) and N-acetyl-DL-norvaline (DL-AcNva) were studied on the basis of thermodynamic analyses to explore the possibility of optical resolution by preferential crystallization.An empirical equation has been derived from thermodynamic data and melting points of ammonium and organic ammonium salts of N-acyl-DL-amino acids to predict racemic structure around room temperature.The AM salts of DL-AcAbu and -AcNva exist in conglomerate around room temperature.It is possible to resolve optically these DL-AM salts by preferential crystallization in ethanol at 10 deg C, and the succesive preferential crystallization followed by purification gave D- and L-2-aminobutyric acids and -norvalines with optical purities close to 100percent.
- Shiraiwa, Tadashi,Yoshida, Hirokazu,Tsuda, Makoto,Kurokawa, Hidemoto
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- Radical SAM Activation of the B12-Independent Glycerol Dehydratase Results in Formation of 5′-Deoxy-5′-(methylthio) adenosine and Not 5′-Deoxyadenosine
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Activation of glycyl radical enzymes (GREs) by S-adenosylmethonine (AdoMet or SAM)-dependent enzymes has long been shown to proceed via the reductive cleavage of SAM. The AdoMet-dependent (or radical SAM) enzymes catalyze this reaction by using a [4Fe-4S] cluster to reductively cleave AdoMet to form a transient 5′deoxyadenosyl radical and methionine. This radical is then transferred to the GRE, and methionine and 5′deoxyadenosine are also formed. In contrast to this paradigm, we demonstrate that generation of a glycyl radical on the B12-independent glycerol dehydratase by the glycerol dehydratase activating enzyme results in formation of 5′deoxy- 5′(methylthio) adenosine and not 5′deoxyadenosine. This demonstrates for the first time that radical SAM activases are also capable of an alternative cleavage pathway for SAM.
- Demick, Jonathan M.,Lanzilotta, William N.
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- Carriebowmide, a new cyclodepsipeptide from the marine cyanobacterium Lyngbya polychroa
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The new cyclodepsipeptide carriebowmide (1), which contains two rare amino acids, 3-amino-2-methylhexanoic acid and methionine sulfoxide, was isolated from the fish-deterrent lipophilic extract of the marine cyanobacterium Lyngbya polychroa, collected from the fore reef near the Smithsonian field station at Carrie Bow Cay, Belize. Its planar structure was determined by NMR spectroscopic techniques. The absolute stereochemistry of the hydroxy acid and all α-amino acid-derived units was ascertained by chiral HPLC analysis of the acid hydrolysate. The stereochemistry of the β-amino acid moiety, 3-amino-2-methylhexanoic acid, was established by Marfey analysis of the acid hydrolysate.
- Gunasekera, Sarath P.,Ritson-Williams, Raphael,Paul, Valerie J.
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- Biocatalytic Cascade Reaction for the Asymmetric Synthesis of L- and D-Homoalanine
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Unnatural amino acids attract growing attention for pharmaceutical applications as they are useful building blocks for the synthesis of a number of chiral drugs. Here, we describe a two-step enzymatic method for the asymmetric synthesis of homoalanine from L-methionine, a cheap and readily available natural amino acid. First, the enzyme L-methionine γ-lyase (METase), from Fusobacterium nucleatum, catalyzed the γ-elimination of L-methionine to 2-oxobutyrate. Second, an amino acid aminotransferase catalyzed the asymmetric conversion of 2-oxobutyrate to either L- or D-homoalanine. The L-branched chain amino acid aminotransferase from Escherichia coli (eBCAT), using L-glutamate as amino donor, produced L-homoalanine (32.5 % conv., 28 % y, 99 % ee) and the D-amino acid aminotransferase from Bacillus sp. (DATA) used D-alanine as amino donor to produce D-homoalanine (87.5 % conv., 69 % y, 90 % ee). Thus, this concept allows for the first time the synthesis of both enantiomers of this important unnatural amino acid.
- Silva, Marcus V. de M.,Costa, Ingrid C. R.,de Souza, Rodrigo O. M. A.,Bornscheuer, Uwe T.
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- Driving Transamination Irreversible by Decomposing Byproduct Α-Ketoglutarate into Ethylene Using Ethylene-Forming Enzyme
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The transformations of transaminases have been extensively studied as an approach to the production of chiral amino moieties. However, the low equilibrium conversion of the reaction is a critical disadvantage to transaminase application, and a strategy for shifting the reaction equilibrium is essential. Herein, we have developed a novel method to effectively prevent the reversibility of transamination by fully decomposing byproduct α-ketoglutarate into ethylene and carbon dioxide in situ using ethylene-forming enzyme (EFE). Two transaminases and one EFE were expressed in E. coli and purified to be used in the cascade reaction. After optimal reaction conditions were determined based on the enzymatic properties, a cascade reaction coupling transaminase with EFE was conducted and showed high efficiency in the synthesis of l-phosphinothricin. Finally, using this approach with only an equivalent amount of amino donor l-glutamate increased the conversions of various keto acids from 99%. This strategy shows great potential for transamination using glutamate as the amino donor.
- Meng, Li-Jun,Liu, Ya-Yun,Zhou, Hai-Sheng,Yin, Xin-Jian,Wu, Jian-Ping,Wu, Mian-Bin,Xu, Gang,Yang, Li-Rong
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- Reductive Cleavage of Sulfoxide and Sulfone by Two Radical S-Adenosyl- l -methionine Enzymes
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Sulfoxides and sulfones are commonly found in nature as a result of thioether oxidation, whereas only a very few enzymes have been found to metabolize these compounds. Utilizing the strong reduction potential of the [4Fe-4S] cluster of radical S-adenosyl-l-methionine (SAM) enzymes, we herein report the first enzyme-catalyzed reductive cleavage of sulfoxide and sulfone. We show two radical SAM enzymes, tryptophan lyase NosL and the class C radical SAM methyltransferase NosN, are able to act on a sulfoxide SAHO and a sulfone SAHO2, both of which are structurally similar to SAM. NosL cleaves all of the three bonds (i.e., S-C(5′), S-C(γ), and S-O) connecting the sulfur center of SAHO, with a preference for S-C(5′) bond cleavage. Similar S-C cleavage activity was also found for SHAO2, but no S-O cleavage was observed. In contrast to NosL, NosN almost exclusively cleaves the S-C(5′) bonds of SAHO and SAHO2 with much higher efficiencies. Our study provides valuable insights into the [4Fe-4S] cluster-mediated reduction reactions and highlights the remarkable catalytic promiscuity of radical SAM enzymes.
- Mandalapu, Dhanaraju,Ji, Xinjian,Zhang, Qi
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- Enantiomeric conversion of racemic amino acid mixtures via an oxidase- aminotransferase coupled system
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D-amino acid oxidase, branched-chain amino acid aminotransferase and excess L-glutamate are used to convert racemic mixtures to the L-form enantiomer in high chemical and optical yields for a number of both common and non-biological amino acids.
- Shah,Schafer,Recchia,Polach,LeMaster
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- Engineering of a novel biochemical pathway for the biosynthesis of L-2- aminobutyric acid in Escherichia coli K12
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L-2-Aminobutyric acid was synthesised in a transamination reaction from L-threonine and L-aspartic acid as substrates in a whole cell biotransformation using recombinant Escherichia coli K12. The cells contained the cloned genes tyrB, ilvA and alsS which respectively encode tyrosine aminotransferase of E. coli, threonine deaminase of E. coli and α- acetolactate synthase of B. subtilis 168. The 2-aminobutyric acid was produced by the action of the aminotransferase on 2-ketobutyrate and L- aspartate. The 2-ketobutyrate is generated in situ from L-threonine by the action of the deaminase, and the pyruvate by-product is eliminated by the acetolactate synthase. The concerted action of the three enzymes offers significant yield and purity advantages over the process using the transaminase alone with an eight to tenfold increase in the ratio of product to the major impurity.
- Fotheringham, Ian G.,Grinter, Nigel,Pantaleone, David P.,Senkpeil, Richard F.,Taylor, Paul P.
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- Rational engineering ofAcinetobacter tandoiiglutamate dehydrogenase for asymmetric synthesis ofl-homoalanine through biocatalytic cascades
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l-Homoalanine, a useful building block for the synthesis of several chiral drugs, is generally synthesized through biocascades using natural amino acids as cheap starting reactants. However, the addition of expensive external cofactors and the low efficiency of leucine dehydrogenases towards the intermediate 2-ketobutyric acid are two major challenges in industrial applications. Herein, a dual cofactor-dependent glutamate dehydrogenase fromAcinetobacter tandoii(AtGluDH) was identified to help make full use of the intracellular pool of cofactors when using whole-cell catalysis. Through reconstruction of the hydrophobic network between the enzyme and the terminal methyl group of the substrate 2-ketobutyric acid, the strict substrate specificity ofAtGluDH towards α-ketoglutarate was successfully changed, and the activity obtained by the most effective mutant (K76L/T180C) was 17.2 times higher than that of the wild-type protein. A three-enzyme co-expression system was successfully constructed in order to help release the mass transfer restriction. Using 1 Ml-threonine, which is close to the solubility limit, we obtained a 99.9% yield ofl-homoalanine in only 3.5 h without adding external coenzymes to the cascade, giving 99.9% ee and a 29.2 g L?1h?1space-time yield. Additionally, the activities of the engineeredAtGluDH towards some other hydrophobic amino acids were also improved to 1.1-11.2 fold. Therefore, the engineering design of some dual cofactor-dependent GluDHs could not only eliminate the low catalytic activity of unnatural substrates but also enhance the cofactor utilization efficiency of these enzymes in industrial applications.
- Diao, Shiqing,Jiang, Shuiqin,Liu, Yan,Sun, Yangyang,Wang, Hualei,Wang, Liuzhu,Wei, Dongzhi
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p. 4208 - 4215
(2021/06/30)
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- Highly Stable Zr(IV)-Based Metal-Organic Frameworks for Chiral Separation in Reversed-Phase Liquid Chromatography
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Separation of racemic mixtures is of great importance and interest in chemistry and pharmacology. Porous materials including metal-organic frameworks (MOFs) have been widely explored as chiral stationary phases (CSPs) in chiral resolution. However, it remains a challenge to develop new CSPs for reversed-phase high-performance liquid chromatography (RP-HPLC), which is the most popular chromatographic mode and accounts for over 90% of all separations. Here we demonstrated for the first time that highly stable Zr-based MOFs can be efficient CSPs for RP-HPLC. By elaborately designing and synthesizing three tetracarboxylate ligands of enantiopure 1,1′-biphenyl-20-crown-6, we prepared three chiral porous Zr(IV)-MOFs with the framework formula [Zr6O4(OH)8(H2O)4(L)2]. They share the same flu topological structure but channels of different sizes and display excellent tolerance to water, acid, and base. Chiral crown ether moieties are periodically aligned within the framework channels, allowing for stereoselective recognition of guest molecules via supramolecular interactions. Under acidic aqueous eluent conditions, the Zr-MOF-packed HPLC columns provide high resolution, selectivity, and durability for the separation of a variety of model racemates, including unprotected and protected amino acids and N-containing drugs, which are comparable to or even superior to several commercial chiral columns for HPLC separation. DFT calculations suggest that the Zr-MOF provides a confined microenvironment for chiral crown ethers that dictates the separation selectivity.
- Jiang, Hong,Yang, Kuiwei,Zhao, Xiangxiang,Zhang, Wenqiang,Liu, Yan,Jiang, Jianwen,Cui, Yong
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supporting information
p. 390 - 398
(2021/01/13)
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- One pot cascade synthesis of L-2-aminobutyric acid employing ω-transaminase from Paracoccus pantotrophus
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ω-transaminase can mediate the asymmetric synthesis of chiral amines from aldehydes and ketones, which has important value in the synthesis of pharmaceutical intermediates. A novel ω-transaminase derived from Paracoccus pantotrophus (PpTA) was obtained and cloned in E. coli BL21(DE3) for expression. The enzyme has high activity for 2-ketobutyric acid and benzylamine, as well as for aromatic compounds with side chains longer than ethyl aliphatic hydrocarbons. Molecular simulation showed that the S-pocket in the active center is larger than those of other ω-transaminases. Thereafter, a whole-cell catalytic system was designed to prepare L-2-aminobutyric acid by cascading PpTA and other enzymes. By using several strategies (regulation of RBS intensity, by-product decomposition and cofactor self-sufficiency), whole-cell cascade biocatalysis showed a high ee value (> 99%) and high yield (71%) in one pot reaction. This study therefore proposes an efficient biocatalyst for the synthesis of unnatural amino acids with the participation of ω-transaminase.
- Dong, Liqing,Hu, Jiagui,Liu, Yuantao,Lu, Jiapeng,Luo, Wei,Wang, Xiaoping,Yu, Xiaobin,Zhang, Huili
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- Robustness, Entrainment, and Hybridization in Dissipative Molecular Networks, and the Origin of Life
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How simple chemical reactions self-assembled into complex, robust networks at the origin of life is unknown. This general problem-self-assembly of dissipative molecular networks-is also important in understanding the growth of complexity from simplicity in molecular and biomolecular systems. Here, we describe how heterogeneity in the composition of a small network of oscillatory organic reactions can sustain (rather than stop) these oscillations, when homogeneity in their composition does not. Specifically, multiple reactants in an amide-forming network sustain oscillation when the environment (here, the space velocity) changes, while homogeneous networks-those with fewer reactants-do not. Remarkably, a mixture of two reactants of different structure-neither of which produces oscillations individually-oscillates when combined. These results demonstrate that molecular heterogeneity present in mixtures of reactants can promote rather than suppress complex behaviors.
- Cafferty, Brian J.,Wong, Albert S. Y.,Semenov, Sergey N.,Belding, Lee,Gmür, Samira,Huck, Wilhelm T. S.,Whitesides, George M.
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supporting information
p. 8289 - 8295
(2019/06/04)
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- Semi-rational hinge engineering: modulating the conformational transformation of glutamate dehydrogenase for enhanced reductive amination activity towards non-natural substrates
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The active site is the common hotspot for rational and semi-rational enzyme activity engineering. However, the active site represents only a small portion of the whole enzyme. Identifying more hotspots other than the active site for enzyme activity engineering should aid in the development of biocatalysts with better catalytic performance. Glutamate dehydrogenases (GluDHs) are promising and environmentally benign biocatalysts for the synthesis of valuable chirall-amino acids by asymmetric reductive amination of α-keto acids. GluDHs contain an inter-domain hinge structure that facilitates dynamic reorientations of the domains relative to each other. Such hinge-bending conformational motions of GluDHs play an important role in regulating the catalytic activity. Thus, the hinge region represents a potential hotspot for catalytic activity engineering for GluDHs. Herein, we report semi-rational activity engineering of GluDHs with the hinge region as the hotspot. Mutants exhibiting significantly improved catalytic activity toward several non-natural substrates were identified and the highest activity increase reached 104-fold. Molecular dynamics simulations revealed that enhanced catalytic activity may arise from improving the open/closed conformational transformation efficiency of the protein with hinge engineering. In the batch production of three valuablel-amino acids, the mutants exhibited significantly improved catalytic efficiency, highlighting their industrial potential. Moreover, the catalytic activity of several active site tailored GluDHs was also increased by hinge engineering, indicating that hinge and active site engineering are compatible. The results show that the hinge region is a promising hotspot for activity engineering of GluDHs and provides a potent alternative for developing high-performance biocatalysts toward chirall-amino acid production.
- Liu, Yayun,Meng, Lijun,Wu, Jianping,Yang, Lirong,Yin, Xinjian,Zhou, Haisheng
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p. 3376 - 3386
(2020/06/09)
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- Enantioselective Synthesis of d- and l-α-Amino Acids by Enzymatic Transamination Using Glutamine as Smart Amine Donor
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Enzymatic transamination is a useful method for the green and highly enantioselective synthesis of chiral amines and non-canonical amino acids which are of major importance as intermediates in medicinal chemistry. However, transamination reactions are usually reversible and synthetic applications of transaminases often require the implementation of an equilibrium shift strategy. Herein, we report a highly effective approach using glutamine as smart amine donor. This amino acid is converted upon transamination into 2-oxoglutaramate which undergoes a fast cyclisation displacing the transamination equilibrium. We have developed a new activity assay in order to identify transaminases from biodiversity able to convert various α-keto acids into valuable amino acids of l- or d-series in the presence of glutamine as amine donor. Discovered transaminases were then used to prepare in high yield and with high enantioselectivity three amino acids of pharmaceutical importance, homophenylalanine, homoalanine and tert-leucine by simply using a nearly stoichiometric amount of glutamine as amine donor. (Figure presented.).
- Heuson, Egon,Charmantray, Franck,Petit, Jean-Louis,de Berardinis, Véronique,Gefflaut, Thierry
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supporting information
p. 778 - 785
(2019/01/04)
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- Continuous-flow protocol for the synthesis of enantiomerically pure intermediates of anti epilepsy and anti tuberculosis active pharmaceutical ingredients
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Continuous-flow production of chiral intermediates plays an important role in the development of building blocks for Active Pharmaceutical Ingredients (APIs), being α-amino acids and their derivatives widely applied as building blocks. In this work we developed two different strategies for the synthesis of intermediates used on the synthesis of levetiracetam/brivaracetam and ethambutol. The results obtained show that methionine methyl ester can be continuously converted to the desired ethambutol intermediate by RANEY Nickel dessulfurization/reduction strategy whereas levetiracetam/brivaracetam intermediates could be synthesized by both RANEY Nickel (without H2) and Pd/C-H2 approach or by photochemical desulfurization.
- Aguiar, Renata M.,Le?o, Raquel A. C.,Mata, Alejandro,Cantillo, David,Kappe, C. Oliver,Miranda, Leandro S. M.,De Souza, Rodrigo O. M. A.
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supporting information
p. 1552 - 1557
(2019/02/14)
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- Combinatorial Mutation Analysis of ω-Transaminase to Create an Engineered Variant Capable of Asymmetric Amination of Isobutyrophenone
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ω-Transaminase (ω-TA) is an important enzyme for asymmetric synthesis of chiral amines. Rapid creation of a desirable ω-TA variant, readily available for scalable process operation, is demanded and has attracted intense research efforts. In this study, we aimed to develop a quantitative mutational analysis (i. e., R-analysis) that enables prediction of combinatorial mutation outcomes and thereby provides reliable guidance of enzyme engineering through combination of already characterized mutations. To this end, we determined three mutatable active-site residues of ω-TA from Ochrobactrum anthropi (i. e., leucine 57, tryptophan 58 and valine 154) by examining activities of nine alanine-scanning mutants for seven substrate pairs. The R-analysis of the mutatable residues is based on assessment of changes in relative activities for a series of structurally analogous substrates. Using three sets of substrates (five α-keto acids, six arylalkylamines and three arylalkyl ketones), we found that combination of two point mutations display additive effects of each mutational outcome such as steric relaxation for bulky substrates or catalytic enhancement for amination of ketones. Consistent with the R-analysis-based prediction, the ω-TA variant harboring triple alanine mutations, i. e. L57A, W58A and V154A, showed high activity improvements for bulky substrates, e. g. a 3.2×104-fold activity increase for 1-phenylbutylamine. The triple mutant even enabled asymmetric amination of isobutyrophenone, carrying a branched-chain alkyl substituent to be accepted in a small binding pocket that normally shows a steric limit up to an ethyl group, with >99% ee of a resulting (S)-amine. (Figure presented.).
- Kim, Hong-Gon,Han, Sang-Woo,Shin, Jong-Shik
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p. 2594 - 2606
(2019/05/15)
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- One-Pot Preparation of d-Amino Acids Through Biocatalytic Deracemization Using Alanine Dehydrogenase and Ω-Transaminase
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d-Amino acids are pharmaceutically important building blocks, leading to a great deal of research efforts to develop cost-effective synthetic methods. Preparation of d-amino acids by deracemization has been conceptually attractive owing to facile synthesis of racemic amino acids by Strecker synthesis. Here, we demonstrated biocatalytic deracemization of aliphatic amino acids into d-enantiomers by running cascade reactions; (1) stereoinversion of l-amino acid to a d-form by amino acid dehydrogenase and ω-transaminase and (2) regeneration of NAD+ by NADH oxidase. Under the cascade reaction conditions containing 100?mM isopropylamine and 1?mM NAD+, complete deracemization of 100?mM dl-alanine was achieved after 24?h with 95% reaction yield of d-alanine (> 99% eeD, 52% isolation yield). Graphical Abstract: [Figure not available: see fulltext.].
- Han, Sang-Woo,Shin, Jong-Shik
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p. 3678 - 3684
(2018/10/20)
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- Bioproduction of l-2-Aminobutyric Acid by a Newly-Isolated Strain of Aspergillus tamarii ZJUT ZQ013
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Abstract: l-2-Aminobutyric acid (l-ABA), an unnatural amino acid, is a key intermediate of several important drugs. Although some methods have been developed to prepare pure chiral l-ABA, there are still many drawbacks, including low catalytic efficiency, cumbersome steps and high cost due to the addition of some expensive catalysts or coenzymes. Herein, with chemical and biological approaches together, we discovered a newly isolated Aspergillus tamarii ZJUT ZQ013 strain containing a microbial lipase which could be employed to resolve racemic methyl N-Boc-2-aminobutyrate to produce l-ABA with high enantioselectivity (e.e.s?>?99.9%, E = 257). Moreover, the subsequent gram scale experiment confrimed that A. tamarii ZJUT ZQ013 could be an attractive biocatalyst for the efficient preparation of optically pure acid. Graphical Abstract: [Figure not available: see fulltext.]
- An, Zhengfang,Gu, Xiaoxu,Liu, Yue,Ge, Jingyan,Zhu, Qing
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p. 837 - 844
(2017/03/24)
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- High-quality S - 2 - butylene-chlorohydrin preparation method
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The invention relates to a preparation method of high-quality S-2-chlorobutanol. The method comprises the following steps: with L-2-aminobutyric acid prepared by a biological reduction transformation method as a raw material, preparing the S-2-chlorobutanol by adopting a diazotization chlorination method; further esterifying, and reducing with sodium borohydride/titanium tetrachloride, so as to obtain the product. According to the prepared high rotary S-2-chlorobutanol, the EE value is over 99%; and the S-2-chlorobutanol is good in repeatability and stable in process.
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Paragraph 0011; 0012; 0013; 0014
(2017/08/25)
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- 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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p. 80557 - 80563
(2016/11/15)
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- METHOD OF PRODUCING D-THREONINE AND HOMOALANINE FROM RACEMIC THREONINE
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Provided is a method for producing D-threonine in a simple way using bio-enzyme. Also, provided is a method for producing homoalanine in high yield from 2-oxobutyrate produced in addition to D-threonine. To this end, the method of the present invention comprises the following steps: (a) producing D-threonine and 2-oxobutyrate from DL-threonine via kinetic resolution involving L-threonine dehydratase; and (b) producing optically pure L-/D-homoalanine using (S)- or (R)-selective omega-transaminase from the 2-oxobutyrate.COPYRIGHT KIPO 2016
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Paragraph 0058-0059
(2017/04/14)
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- Preparation of d-threonine by biocatalytic kinetic resolution
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D-Threonine is one of the important unnatural amino acids used as chiral building blocks in pharmaceutical drugs. Owing to the presence of two chiral centers, a synthetic protocol, either through chemocatalysis or biocatalysis, has not yet been available for one-step preparation of stereochemically pure d-threonine in terms of enantiomeric and diastereomeric excesses (i.e., both >99%). Here we demonstrate that facile production of d-threonine can be implemented using threonine deaminase (TD) via kinetic resolution of dl-threonine that can be readily prepared by conventional organic synthesis. TD catalyzes the dehydration/deamination of l-threonine, leading to generation of 2-oxobutyrate and ammonia. In contrast to mild substrate inhibition of the TD activity by l-threonine (i.e., apparent inhibition constant (KIapp) = 950 mM), d-threonine turned out to be a strong inhibitor (i.e., KIapp = 41 mM). In addition to the enzyme inhibitions by both enantiomers of threonine, cell lysis observed during small-scale kinetic resolutions of ≥1 M dl-threonine led us to carry out a preparative-scale reaction at 500 mM racemic substrate. The preparative-scale kinetic resolution in a 50 mL reaction mixture charged with 3 g dl-threonine and 3400 U whole cells was completed at 5 h with >99% ee of d-threonine. Product isolation by a cation-exchange chromatography led to white solid of d-threonine (1.36 g, 90.7% isolation yield). To explore whether our strategy could afford coproduction of another valuable unnatural amino acid, the pass-through solution from the cation-exchange column was further processed by a ω-transaminase (ω-TA) reaction where 2-oxobutyrate was converted to enantiopure homoalanine using isopropylamine as an amino donor. Addition of S- and R-selective ω-TA to the pass-through solution led to 93.2 and 90.9% reaction yield within 12 h with both >99% ee of the produced l- and d-homoalanine, respectively.
- Han, Sang-Woo,Shin, Jong-Shik
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p. 227 - 232
(2015/10/28)
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- Preparation method of optically active amines and homoalanine
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The present invention refers to optically active relates to manufacturing method of a chiral amine. The present invention according to manufacturing method whereby an optical active chiral for producing an amine when the low-cost amino receptor by using production thereof in the preparation of optically active chiral amine USB data switches a, such low-cost amino receptor using primer even optically active excellent manufacturing efficiency of a chiral amine.. In addition single enantiomers of is connected with a, furthermore said manufacturing method is naturally through the file does not exist at L- [...] production and..
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Paragraph 0048
(2017/01/02)
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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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- Amidohydrolase Process: Expanding the use of l-N-carbamoylase/N-succinyl- amino acid racemase tandem for the production of different optically pure l-amino acids
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A bienzymatic system comprising an N-succinylamino acid racemase from Geobacillus kaustophilus CECT4264 (GkNSAAR) and an enantiospecific l-N-carbamoylase from Geobacillus stearothermophilus CECT43 (BsLcar) has been developed. This biocatalyst has been able to produce optically pure natural and non-natural l-amino acids starting from racemic mixtures of N-acetyl-, N-formyl- and N-carbamoyl-amino acids by dynamic kinetic resolution. The fastest conversion rate was found with N-formyl-amino acids, followed by N-carbamoyl- and N-acetyl-amino acids, and GkNSAAR proved to be the limiting step of the system due to its lower specific activity. Metal ion cobalt was essential for the activity of the biocatalyst and the system was optimally active when Co 2+ was added directly to the reaction mixture. The optimum pH for the biocatalyst proved to be 8.0, for both N-formyl- and N-carbamoyl-amino acid substrates, whereas optimum temperature ranges were 45-55 °C for N-formyl-amino acids and 55-70 °C for N-carbamoyl-derivatives. The bienzymatic system was equally efficient in converting aromatic and aliphatic substrates. Total conversion was also achieved using high substrate concentrations (100 and 500 mM) with no noticeable inhibition. This "Amidohydrolase Process" enables the production of both natural and non-natural l-amino acids from a broad substrate spectrum with yields of over 95%.
- Soriano-Maldonado, Pablo,Rodríguez-Alonso, María José,Hernández-Cervantes, Carmen,Rodríguez-García, Ignacio,Clemente-Jiménez, Josefa María,Rodríguez-Vico, Felipe,Martínez-Rodríguez, Sergio,Las Heras-Vázquez, Francisco Javier
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p. 1281 - 1287
(2014/07/22)
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- Deracemization of amino acids by coupling transaminases of opposite stereoselectivity
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Biocatalytic deracemization of amino acids without relying on oxidase-based deamination of an unwanted enantiomer was demonstrated by coupling a-and w-transaminases displaying opposite stereoselectivity. This strategy employs isopropylamine and a keto acid as cosubstrates and is free of generation of hydrogen peroxide which is troublesome in the conventional oxidase-based methods.
- Park, Eul-Soo,Shin, Jong-Shik
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p. 3505 - 3509
(2015/02/19)
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- ω-Transaminase-catalyzed asymmetric synthesis of unnatural amino acids using isopropylamine as an amino donor
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Isopropylamine is an ideal amino donor for reductive amination of carbonyl compounds by ω-transaminase (ω-TA) owing to its cheapness and high volatility of a ketone product. Here we developed asymmetric synthesis of unnatural amino acids via ω-TA-catalyzed amino group transfer between α-keto acids and isopropylamine.
- Park, Eul-Soo,Dong, Joo-Young,Shin, Jong-Shik
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p. 6929 - 6933
(2013/10/08)
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- Biocatalytic asymmetric synthesis of unnatural amino acids through the cascade transfer of amino groups from primary amines onto keto acids
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Flee to the hills: An unfavorable equilibrium in the amino group transfer between amino acids and keto acids catalyzed by α-transaminases was successfully overcome by coupling with a ω-transaminase reaction as an equilibrium shifter, leading to efficient asymmetric synthesis of diverse unnatural amino acids, including L-tert-leucine and D-phenylglycine. Copyright
- Park, Eul-Soo,Dong, Joo-Young,Shin, Jong-Shik
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p. 3538 - 3542
(2014/01/06)
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- ω-Transaminase-catalyzed kinetic resolution of chiral amines using l-threonine as an amino acceptor precursor
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Kinetic resolution of chiral amines using l-threonine as a cosubstrate was demonstrated by a biocatalytic strategy in which (S)-selective ω-transaminase (ω-TA) was coupled with threonine deaminase (TD), eliminating the need to use an expensive keto acid as an amino acceptor. The coupled enzyme reaction enabled simultaneous production of enantiopure (R)-amine and l-homoalanine which are pharmaceutically important building blocks. To extend the versatility of this strategy to production of both enantiomers of chiral amines, (R)-selective ω-TA coupled with TD was employed to produce (S)-amine.
- Malik, M. Shaheer,Park, Eul-Soo,Shin, Jong-Shik
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supporting information; experimental part
p. 2137 - 2140
(2012/09/25)
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- Dynamic kinetic resolution of α-aminonitriles to form chiral α-amino acids
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We have succeeded in the enzymatic synthesis of (R)-α-aminobutyric acid from racemic α-aminobutyronitrile. This has been demonstrated by the use of non-stereoselective nitrile hydratase (NHase) from Rhodococcus opacus 71D, D-aminopeptidase from Ochrobactrum anthropi C1-38 and α-amino-ε- caprolactam (ACL) racemase from Achromobacter obae. Racemic α- aminobutyronitrile was completely converted in 6 h at 30 °C to (R)-α-aminobutyric acid whose optical purity was more than 99%. (S)-α-Aminobutyric acid was also synthesized from α- aminobutyronitrile by NHase, ACL racemase and L-amino acid amidase from Brevundimonas diminuta TPU 5720. In a similar manner, other (R)- or (S)-α-amino acids with more than 97.5% ee could be synthesized from the corresponding α-aminonitriles. This is the first report on the dynamic kinetic resolution (DKR) of α-aminonitriles to form chiral α-amino acids. The key enzyme in this DKR is non-stereoselective NHase, which had been newly screened from soil samples, and its gene cloned. Copyright
- Yasukawa, Kazuyuki,Hasemi, Ryuji,Asano, Yasuhisa
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scheme or table
p. 2328 - 2332
(2011/10/19)
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- One-pot conversion of L -threonine into L -homoalanine: Biocatalytic production of an unnatural amino acid from a natural one
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A novel biocatalytic process for production of L-homoalanine from L-threonine has been developed using coupled enzyme reactions consisting of a threonine deaminase (TD) and an ω-transaminase (ω-TA). TD catalyzes the dehydration/deamination of L-threonine, leading to the generation of 2-oxobutyrate which is asymmetrically converted to L-homoalanine via transamination with benzylamine executed by ω-TA. To make up the coupled reaction system, we cloned and overexpressed a TD from Escherichia coli and an (S)-specific ω-TA from Paracoccus denitrificans. In the coupled reactions, L-threonine serves as a precursor of 2-oxobutyrate for the ω-TA reaction, eliminating the need for employing the expensive oxo acid as a starting reactant. In contrast to α-transaminase reactions in which use of amino acids as an exclusive amino donor limits complete conversion, amines are exploited in the ω-TA reaction and thus maximum conversion could reach 100%. The ω-TA-only reaction with 10 mM 2-oxobutyrate and 20 mM benzylamine resulted in 94% yield of optically pure L-homoalanine (ee>99%). However, the ω-TA-only reaction did not produce any detectable amount of L-homoalanine from 10 mM L-threonine and 20 mM benzylamine, whereas the ω-TA reaction coupled with TD led to 91% conversion of L-threonine to L-homoalanine. Copyright
- Park, Eulsoo,Kim, Minji,Shin, Jong-Shik
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scheme or table
p. 3391 - 3398
(2011/02/23)
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- METHOD OF SEPARATING AND COLLECTING OPTICALLY ACTIVE AMINO ACID AMIDE
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[PROBLEMS] To provide a method of efficiently separating and collecting an optically active amino acid amide and an optically active amino acid, which are highly important substances as intermediates in producing various industrial products, pesticides and medicines, from an aqueous solution containing the optically active amino acid amide and the optically active amino acid. [MEANS FOR SOLVING PROBLEMS] In separating and collecting an optically active amino acid amide from an aqueous solution containing the optically active amino acid amide and an optically active amino acid by using the difference in solubility in an organic solvent between the optically active amino acid amide and the optically active amino acid, the separation/collection procedures are carried out, without desalting the aqueous solution or after desalting the same, under such conditions that the ratio (C/A) of the sum of the anionic equivalents (A) contained in the aqueous solution to the sum of the cation equivalents (C) ranges from 0.95 to 1.05 in the case of the non-desalted aqueous solution, or from 0.5 to 1.5 in the case of the desalted aqueous solution.
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Page/Page column 11-12;13
(2008/06/13)
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- Synthesis of α-amino acids by reaction of aziridine-2-carboxylic acids with carbon nucleophiles
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A variety of homochiral α-amino acids have been prepared in good yield via regioselective reaction of higher order cuprates with (2S)-N-para-toluenesulfonylaziridine-2-carboxylic acid 4. The reaction was much less regioselective and low yielding when higher order cuprates were reacted with the more hindered aziridine carboxylic acid 30, the principal products being protected β-amino acids. Reaction of lithium trimethylsilylacetylide with the aziridine acid 30, however, gave a protected α-amino acid which was converted to the protected isoleucine ester 37. The Royal Society of Chemistry 2006.
- Beresford, Kenneth J. M.,Church, Nicola J.,Young, Douglas W.
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p. 2888 - 2897
(2008/02/08)
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- Resolution of non-protein amino acids via the microbial protease-catalyzed enantioselective hydrolysis of their N-unprotected esters
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In the Aspergillus oryzae protease-catalyzed ester hydrolysis, substitution of N-unprotected amino acid esters for the corresponding N-protected amino acid esters resulted in a large enhancement of the hydrolysis rate, while the enantioselectivity was deteriorated strikingly when the substrates employed were the conventional methyl esters. This difficulty was overcome by employing esters bearing a longer alkyl chain such as the isobutyl ester. Utilizing this ester, amino acids carrying an aromatic side chain were resolved with excellent enantioselectivities (E=50 to >200). With amino acids bearing an aliphatic side chain also, good results in terms of the hydrolysis rate and enantioselectivity were obtained by employing such an ester as the isobutyl ester. Moreover, the enantioselectivity proved to be enhanced further by conducting the reaction at low temperature. This procedure was applicable to the case where the enantioselectivity was not high enough even by the use of the isobutyl ester.
- Miyazawa, Toshifumi,Imagawa, Kiwamu,Minowa, Hiroe,Miyamoto, Toyoko,Yamada, Takashi
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p. 10254 - 10261
(2007/10/03)
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- Application of aminoacylase I to the enantioselective resolution of α-amino acid esters and amides
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Aminoacylase I from Aspergillus melleus, a readily available and inexpensive enzyme mainly used in the industrial production of enantiopure L-amino acids from their N-acetyl derivatives, is shown to hydrolyze the esters and amides of natural and non-natural amino acids with high enantioselectivity (for the ester hydrolysis, E is up to 76, in case of amides E >300). The reaction rates of amide and ester hydrolysis are comparable, and in some cases these conversions proceeded even faster than 'traditional' aminoacylase- catalyzed hydrolysis of N-acetyl derivatives thus providing new possibilities for the resolution of the corresponding racemates. This novel approach provides an alternative route for the biocatalytic production of optically active amino acids and their derivatives.
- Youshko, Maxim I.,Van Langen, Luuk M.,Sheldon, Roger A.,Svedas, Vytas K.
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p. 1933 - 1936
(2007/10/03)
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- Asymmetric Strecker synthesis by addition of trimethylsilyl cyanide to aldehyde SAMP-hydrazones
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The asymmetric 1,2-addition of trimethylsilyl cyanide to aldehyde SAMP-hydrazones in the presence of titanium tetrachloride and diethylether in dichloromethane at -100°C up to room temperature, removal of the chiral auxiliary and acid hydrolysis affords α-amino acids in high enantiomeric excesses (ee=94-97%).
- Enders, Dieter,Moser, Michael
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p. 8479 - 8481
(2007/10/03)
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- Deracemisation and stereoinversion of alpha-amino acids using D-amino acid oxidase and hydride reducing agents.
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The deracemisation and stereoinversion of both cyclic and acyclic DL-alpha-amino acids, using porcine kidney D-amino acid oxidase (DAAO) and a hydride reducing agent (NaCNBH3-NaBH4), has been investigated.
- Beard, Timothy M,Turner, Nicholas J
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p. 246 - 247
(2007/10/03)
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- Highly efficient catalytic synthesis of α-amino acids under phase-transfer conditions with a novel catalyst/substrate pair
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A facile and fast enantioselective synthesis of α-amino acids with high ee values was achieved by the asymmetric alkylation of the glycine derivative 1 under phase-transfer conditions with (R)-2-amino-2′-hydrozy-1,1′-binaphthyl (NOBIN; see sceme). The ee value of the amino acid products. This occures as a results of a significant positive nonlinear effect in the alkylation reaction.
- Belokon, Yuri N.,Kochetkov, Konstantin A.,Churkina, Tatiana D.,Ikonnikov, Nikolai S.,Larionov, Oleg V.,Harutyunyan, Syuzanna R.,Vyskocil, Stepan,North, Michael,Kagan, Henri B.
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p. 1948 - 1951
(2007/10/03)
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- The imine (+)-pseudoephedrine glycinamide: A useful reagent for the asymmetric synthesis of (R)-α-amino acids
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The new imine derived from Myers (+)-pseudoephedrine glycinamide can be diastereoselectively alkylated with alkyl halides at room temperature using NaOEt or LiO-tert-Bu as bases under phase transfer conditions. Hydrolysis to the corresponding alkylated products was easily achieved under mild conditions to afford (R)-α-amino acids.
- Guillena, Gabriela,Najera, Carmen
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p. 181 - 183
(2007/10/03)
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- Dynamic kinetic resolution of racemic N-phthalyl amino acids using (S)-α-methylpantolactone as the chiral auxiliary
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A dynamic kinetic resolution of racemic N-phthalyl amino acids by stereoselective esterification was examined using (S)-α-methylpantolactone as the chiral auxiliary. The reaction of various racemic N-phthalyl amino acids with this chiral alcohol in the presence of both DCC and DMAP afforded predominantly the (S,S)-esters in nearly quantitative yield. Copyright (C) 2000 Elsevier Science Ltd.
- Calmes, Monique,Glot, Christele,Michel, Thibaut,Rolland, Marc,Martinez, Jean
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p. 737 - 741
(2007/10/03)
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- Chemo-enzymatic synthesis of optically active amino acids and peptides
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The industrial alkaline protease, alcalase, is stable and active in a high concentration of organic solvents and useful as a biocatalyst for (i) diastereoselective hydrolysis of peptide esters and preparation of racemization-free peptides; (ii) selective incorporation of esters of D-amino acid into peptides in t-butanol via a selective hydrolysis of esters of D,L-amino acid, followed by using the unhydrolyzed D-esters as a nucleophile in a kinetically controlled peptide bond formation; (iii) resolution of esters of amino acid in 95% t-butanol/5% water, followed by saponification of the unreacted esters to offer both enantiomers with high yield and optical purity; (iv) completely resolve amino-acid esters with high yield and optical purity via in situ racemization of the unreacted antipode catalyzed by pyridoxal 5-phosphate; (v) cryobioorganic synthesis of peptides with increased yields 15%-40% of peptide bond formation by reaction at 5 °C instead of 25-30 °C of a kinetically controlled enzymatic reaction in alcohols.
- Chen, Shui-Tein,Wang, Kung-Tsung
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p. 301 - 311
(2007/10/03)
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- Stereospecificity of Pseudomonas fluorescens kynureninase for diastereomers of β-methylkynurenine
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The diastereomers of β-methyl-L-kynurenine were prepared by preparative ozonolysis of the respective diastereomers of β-methyl-L-tryptophan. A practical method for preparative enzymatic resolution of the diastereomers of β-methyltryptophan was developed using carboxypeptidase A digestion of the N-trifluoroacetyl derivatives. The stereochemical assignment was confirmed by X-ray crystal structure determination of (2S,3R)-threo-β-methyl-L-tryptophan. (2S,3S)-erythro-β-Methyl-L-kynurenine is a slow substrate for kynureninase from Pseudomonas fluorescens (k(cat)/K(m)=0.1% that of L-kynurenine), producing anthranilic acid, while (2S,3R)-threo-L-kynurenine is about 390-fold less reactive than erythro. Rapid-scanning stopped-flow measurements show that β-methyl substitution affects the rate of α-deprotonation of the L-kynurenine-pyridoxal-5'-phosphate Schiff's base. This is consistent with the stereoelectronic requirements of the reaction. These results are the first demonstration that β-substituted kynurenines can be substrates for kynureninase, and may be useful in the design of mechanism-based inhibitors. Copyright (C) 1999 Elsevier Science Ltd.
- Cyr, Lakshmi V.,Newton, M. Gary,Phillips, Robert S.
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p. 1497 - 1503
(2007/10/03)
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- Flame-induced reactions of sulfur-containing amino acids in an aqueous solution
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Hydrogen-oxygen flames, when blown against an aqueous solution of methionine, induced conversion reactions to homoserine, 2-aminobutyric acid and glutamic acid. Besides the already-known reactions by a hydroxyl radical, a contribution of a hydrogen atom from hydrogen-rich flames to the reaction was recognized. We successfully controlled the vigorous oxidation of the system using a radical scavenger.
- Nomoto, Shinya,Shimoyama, Akira,Shiraishi, Susumu
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p. 1009 - 1012
(2007/10/03)
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- Synthesis of enantiomerically pure α-amino acids via chemo- and diastereoselective alkylation of (5S)-5-phenyl-5,6-dihydro-2H-1,4-oxazin-2-one
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(5S)-5-Phenyl-5,6-dihydro-2H-1,4-oxazin-2-one 2 undergoes Lewis acid-mediated chemo- and diastereoselective nucleophilic addition of Grignard reagents to furnish adducts 3 which can be dismantled to allow ready access to enantiomerically pure (S)-α-amino acids 4.
- Harwood, Laurence M.,Tyler, Simon N. G.,Anslow, A. Susan,MacGilp, Iain D.,Drew, Michael G. B.
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p. 4007 - 4010
(2007/10/03)
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- L-Methionine related 1-amino acids by acylase cleavage of their corresponding N-acetyl-DL-derivatives
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Acylase I from Aspergillus oryzae is an even more useful enzyme than suggested so far. Besides standard amino acids such as L-Met, L-Val and L-Phe, a number of additional sulfur- and selenium-containing amino acids can be obtained at useful reaction rates and in very high enantiomeric purity by kinetic resolution of the respective N-acetyl-DL-amino acids.
- Bommarius, Andreas S.,Drauz, Karlheinz,Guenther, Kurt,Knaup, Guenter,Schwarm, Michael
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p. 3197 - 3200
(2007/10/03)
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- Asymmetric synthesis of α-amino acids via diastereoselective addition of (R)-pantolactone to their ketenes
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The diastereoselective addition of (R)-pantolactone to various amino ketenes derived from phthalylamino acids is reported. The configuration of the newly-generated asymmetric center is dependent on alkyl or aryl C(x substitution. This method constitutes a novel and convenient way of amino acid deracemization.
- Calmes, Monique,Daunis, Jacques,Mai, Nathalie
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p. 1641 - 1648
(2007/10/03)
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- Resolution of non-protein amino acids via microbial protease-catalyzed ester hydrolysis: Marked enhancement of enantioselectivity by the use of esters with longer alkyl chains and at low temperature
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In the microbial protease-catalyzed hydrolysis of amino acid esters with the free α-amino group, the enantioselectivity can be enhanced greatly by employing esters with longer alkyl chains such as the isobutyl ester instead of the conventional methyl ester and by conducting the reaction at low temperature.
- Miyazawa, Toshifumi,Minowa, Hiroe,Miyamoto, Toyoko,Imagawa, Kiwamu,Yanagihara, Ryoji,Yamada, Takashi
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p. 367 - 370
(2007/10/03)
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