- Molecular Structure of a Chiral 3,5-Bridged Pyridine and the Effect of Structure on Circular Dichroic Spectra
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The crystal structure of the 3,5-bridged chiral macrocyclic pyridine (4S,14S)-4,14-di(2-propyl)-6,9,12-trioxa-3,15,19-triazabicycloheneicosa-1(21),17,19-triene-2,5,13,16-tetrone (5a) has been determined by crystallographic means.Each unit cell contains two nonequivalent molecules.In each molecule the amide groups are twisted out-of-plane in a conrotatory fashion righ-handedly with respect to the molecular C2 axis viewed along the line from C4 to N1 of the pyridine ring.This twist allows avoidance of potential interaction between the amide nitrogen bonded protons and that bonded to C4 of the pyridine ring.The macrocyclic framework is inherently dissymmetric as a result of this helical twist.This is reflected in the circular dichroism spectrum of 5a, which has two strongly negative effects in the 200-400-nm region, at 218 nm, -58800 and 273 nm, -45600.Very similar CD effects are found for analogues of 5a with at the chiral atoms at the 4,14-positions, methyl groups (6a), tert-butyl groups (6b), and proline (7).Comparison are also made with compounds (8b) derived (in thought) from 5a by transposition of the macrocyclic bridge from the 3,5- to the 2,6-positions.Compound 8a is analogous to 8b save that it is a benzene rather than a pyridine derivative.Several nonmacrocyclic analogues of 5a have also been examined as well as the thiamide derivative of 5a (compound 9) for which a synthesis has been developed.The longer wavelength CD effect in 5a is assigned to the pyridine n-?* transition and the shorter wavelength effect to ?-?* transitions.Attempts to correlate the absolute signs with a recently postulated model fail.A method for synthesis of the unnatural amino acids, (S)-(+)-2-amino-3,3,-dimethylbutanoic acid (13), in enantiomerically pure form is described as well as an NMR method for the determination of the enantiomeric purity of samples of 13.
- Speelman, Johanna C.,Talma, Auke G.,Kellogg, Richard M.,Meetsma, A.,Boer, J. L. de,et al.
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- Design of a self-sufficient hydride-shuttling cascade for concurrent bioproduction of 7,12-dioxolithocholate andl-tert-leucine
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Oxidoreductase-mediated biotransformation often requires consumption of a secondary sacrificial co-substrate and an additional auxiliary enzyme to drive the cofactor regeneration, which results in generation of unwanted by-product. Herein, we report a highly atom-economic self-sufficient hydride-shuttling cascade to concurrently obtain two pharmaceutically important building blocks (7,12-dioxo-lithocholic acid andl-tert-leucine) in which oxidation of cholic acid (CA) and reductive amination of trimethylpyruvic acid were integrated for redox self-recycling. In this cascade, the cofactor acts as a hydride shuttle that interconnects the two synthetically relevant reactions at the cost of only inorganic ammonium as the sacrificial agent and generates water as the greenest by-product. The preparative biotransformation using a whole-cell biocatalyst in the absence of any exogenous cofactor displayed a space-time yield of 768 g L?1d?1and a total turnover number (TTN) of 20?363 for NAD+recycling. This represents the highest cofactor TTN reported to date for the bio-oxidation of CA, indicating the great potential of this cofactor and redox self-sufficient bioprocess for cost-effective and sustainable biomanufacturing of high-value-added products.
- Chen, Qi,Han, Yu,Li, Chun-Xiu,Pan, Jiang,Qian, Xiao-Long,Xu, Jian-He,Yang, Bing-Yi,You, Zhi-Neng,Zhou, Ke
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- SIMPLE OPTICAL RESOLUTION OF TERLEUCINE
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Underivatized terleucine (1) can be conveniently resolved into its L- and D-enantiomers by recrystallization of its diastereoisomeric 10-camphorsulphonate salts.
- Viret, Joelle,Patzelt, Heiko,Collet, Andre
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- Asymmetric synthesis of l-6-hydroxynorleucine from 2-keto-6-hydroxyhexanoic acid using a branched-chain aminotransferase
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l-6-Hydroxynorleucine was synthesized from 2-keto-6-hydroxyhexanoic acid using branched-chain aminotransferase from Escherichia coli with l-glutamate as an amino donor. Since the branched-chain aminotransferase was severely inhibited by 2-ketoglutarate, the branched-chain aminotransferase reaction was coupled with aspartate aminotransferase and pyruvate decarboxylase. Aspartate aminotransferase converted the inhibitory 2-ketoglutarate back to l-glutamate by using l-aspartate as an amino donor. On the other hand, pyruvate decarboxylase further shifted the reaction equilibrium towards l-6-hydroxynorleucine through decarboxylation of pyruvate to acetaldehyde. The concerted action of the three enzymes significantly enhanced the yield compared to that of branched-chain aminotransferase alone. In the coupled reaction, 90.2 mM l-6-hydroxynorleucine (> 99% ee) was produced from 100 mM 2-keto-6-hydroxyhexanoic acid, whereas in a single branched-chain aminotransferase reaction only 22.5 mM l-6-hydroxynorleucine (> 99% ee) was produced.
- Seo, Young-Man,Kim, Aran,Bea, Han-Seop,Lee, Sang-Hyeup,Yun, Hyungdon
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- Cadmium sulfide net framework nanoparticles for photo-catalyzed cell redox
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A strategy for synthesizing cadmium sulfide net framework (CdS-NF) nanoparticles was developed in a water-based system under mild reaction conditions. The CdS-NFs have not only the excellent photocatalytic properties of CdS, but also the large surface area and diverse porous structures of a metal-organic framework. An Escherichia coli-CdS-NF hybrid system was constructed using NADH regeneration to promote the conversion of trimethylpyruvate acid to l-tert-leucine. The E. coli-CdS-NF system showed higher NAD+ recycling efficiency and substrate conversion rate than CdS QDs under visible light illumination. This work demonstrates a novel method for developing a brilliant coenzyme recycling photocatalyst in bio-redox reactions.
- Chang, Zhaoyu,Dong, Wanyuan,Meng, Xiangqi,Ren, Yuhong,Wang, Hualei,Wei, Dongzhi,Zhang, Jian
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- Biocatalytic cascade reactions for asymmetric synthesis of aliphatic amino acids in a biphasic reaction system
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Abstract Enantiopure aliphatic amino acids, including l-3-hydroxyadamantylglycine (l-Hag), l-tert-leucine (l-Tle) and l-norvaline, are essential chiral building blocks for a number of pharmaceutical drugs. Here, we developed cascade enzyme reactions in an extractive biphasic system using a branched-chain amino acid transaminase (BCTA) and an (S)-selective ω-transaminase (ω-TA) for asymmetric synthesis of the aliphatic amino acids from achiral α-keto acid precursors. The extractive cascade reactions enabled equilibrium shift of the BCTA reaction by recycling an amino acid cosubstrate as well as acceleration of the ω-TA reaction by removing an inhibitory ketone product from an aqueous phase. Starting with 20 mM α-keto acid, 4 mM rac-homoalanine and 50 mM rac-α-methylbenzylamine (rac-α-MBA), the biphasic cascade reactions afforded synthesis of four unnatural amino acids (i.e., l-Tle, l-Hag, l-norvaline and l-norleucine) and two natural amino acids (i.e., l-valine and l-Leucine) with >92% conversion yield and >99.9% ee. To demonstrate the industrial feasibility of the extractive cascade reaction, preparative-scale synthesis of l-Hag was performed in a reaction mixture consisting of 300 mL hexane and 50 mL aqueous solution (50 mM phosphate buffer, pH 7.0) charged with 50 mM keto acid substrate, 5 mM l-homoalanine, 120 mM rac-α-MBA, 2 U/mL BCTA and 16 U/mL ω-TA. Conversion yield of l-Hag reached 92% with >99.9% ee at 70 h. Product isolation led to 0.32 g white solid of l-Hag (62 % isolation yield).
- Park, Eul-Soo,Shin, Jong-Shik
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- Formate Dehydrogenase from Rhodococcus jostii (RjFDH) – A High-Performance Tool for NADH Regeneration
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The use of formate dehydrogenases (FDHs) for regeneration of the important cofactor NADH in enzyme-catalysed synthetic reactions has several advantages over alternative systems. However, a major bottleneck for broad industrial applications is the low specific activity of the currently used FDHs. In this study, we introduce a novel NAD-dependent formate dehydrogenase from Rhodococcus jostii (RjFDH) with both high specific activity and stability. The enzyme was identified in a targeted database research and recombinantly obtained from Escherichia coli. RjFDH is a homodimer with a monomeric molecular mass of 44.7 kDa. The homology model shows that all amino acid residues of the NAD-dependent formate dehydrogenases are usually concerned with catalytic activity, substrate acceptance, and cofactor binding. The only substrate oxidised by these enzymes is formate. RjFDH had a specific activity of 19.9 U mg?1 at 22 °C along with unimpaired activity and high stability over a broad pH range. The Km values for formate and NAD+ were 7.3 and 0.098 mmol L?1, respectively. The optimum temperature was found to be 50 °C, at which the enzyme activity increased to about 318%. Both activity and thermal stability were higher than those of the FDH from Candida boidinii (CbFDH), which is the standard enzyme currently in use for cofactor regeneration. Different solvents roughly had the same impact on the activity and stability of both RjFDH and CbFDH. The superior performance of RjFDH over CbFDH as a regeneration system for NADH was demonstrated for the synthesis of L-tert-leucine as well as (S)-1-phenylethanol. In both systems, the concentration of RjFDH used was only one-third of the concentration of CbFDH required to achieve comparable conversion rates. Rational designing provided a promising NADP-accepting variant. Thus, RjFDH has a great potential to serve as an alternative system for NADH regeneration in enzyme-catalysed synthetic reactions. (Figure presented.).
- Boldt, Alexander,Ansorge-Schumacher, Marion B.
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- Artificial multienzyme supramolecular device: Highly ordered self-assembly of oligomeric enzymes in vitro and in vivo
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A strategy for scaffold-free self-assembly of multiple oligomeric enzymes was developed by exploiting enzyme oligomerization and protein-protein interaction properties, and was tested both in vitro and in vivo. Octameric leucine dehydrogenase and dimeric formate dehydrogenase were fused to a PDZ (PSD95/Dlg1/zo-1) domain and its ligand, respectively. The fusion proteins self-assembled into extended supramolecular interaction networks. Scanning-electron and atomic-force microscopy showed that the assemblies assumed two-dimensional layer-like structures. A fluorescence complementation assay indicated that the assemblies were localized to the poles of cells. Moreover, both in vitro and in vivo assemblies showed higher NAD(H) recycling efficiency and structural stability than did unassembled structures when applied to a coenzyme recycling system. This work provides a novel method for developing artificial multienzyme supramolecular devices and for compartmentalizing metabolic enzyme cascades in living cells.
- Gao, Xin,Yang, Shuai,Zhao, Chengcheng,Ren, Yuhong,Wei, Dongzhi
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- Synthesis method of L-tertiary leucine and L-cyclohexyl alanine
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The invention discloses a synthesis method of L-tertiary leucine and L-cyclohexyl alanine, and belongs to the technical field of amino acid preparation. The method comprises the following steps: taking pentafluorophenol-(dibenzylamine) ester 1 and benzhydrol 2 as reactants, carrying out dynamic kinetic resolution reaction under the catalysis of chiral PPY nitrogen oxygen 3 to obtain a compound 4, and then carrying out Pd/C catalytic hydrogenation debenzylation to obtain L-tertiary leucine and L-cyclohexyl alanine. The oxygen atom in pyridine nitrogen oxygen in the chiral PPY nitrogen oxygen catalyst is used as a nucleophilic site to participate in dynamic kinetic resolution. The method has the advantages of being good in yield, high in enantioselectivity and the like, the ee of the obtained product can reach 99.5% or above, and a new path is provided for synthesis of chiral amino acid.
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Paragraph 0030-0032
(2021/08/07)
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- A Transient Directing Group Strategy Enables Enantioselective Multicomponent Organofluorine Synthesis
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The vicinal fluorofunctionalization of alkenes represents an expedient strategy for converting feedstock olefins into valuable fluorinated molecules and as such has garnered significant attention from the synthetic community; however, current methods remain limited in terms of scope and selectivity. Here we report the site-selective palladium-catalyzed three-component coupling of alkenylbenzaldehydes, arylboronic acids, and N-fluoro-2,4,6-trimethylpyridinium hexafluorophosphate facilitated by a transient directing group. The synthetically enabling methodology constructs vicinal stereocenters with excellent regio-, diastereo-, and enantioselectivities, forging products that map onto bioactive compounds.
- Engle, Keary M.,Gao, Yang,Li, Zi-Qi,Liu, Mingyu,Liu, Zhonglin,Oxtoby, Lucas J.,Tran, Van T.
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supporting information
p. 8962 - 8969
(2021/07/01)
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- Method for preparing D-type or L-type tert-leucine
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The invention discloses a method for preparing D-type or L-type tert-leucine, and belongs to the technical field of organic synthesis. The method comprises the following steps: 1, condensing glyoxylate serving as a raw material with chiral tert-butyl sulfinamide to obtain Schiff base; and 2, carrying out a reaction on the Schiff base with a tert-butyl Grignard reagent and a catalyst under a low-temperature condition, and hydrolyzing the reaction product under an acid/alkali condition to obtain D-type or L-type tert-leucine. The method is simple and convenient to operate and high in reaction yield, the purity and content of the obtained product are greater than 99%, the content of a single impurity is less than 0.2%, the chiral purity is greater than or equal to 98.5%, and the method has apotential process amplification prospect.
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Paragraph 0036; 0040-0042
(2020/06/16)
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- Bioelectrocatalytic Conversion from N2 to Chiral Amino Acids in a H2/α-Keto Acid Enzymatic Fuel Cell
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Enzymatic electrosynthesis is a promising approach to produce useful chemicals with the requirement of external electrical energy input. Enzymatic fuel cells (EFCs) are devices to convert chemical energy to electrical energy via the oxidation of fuel at the anode and usually the reduction of oxygen or peroxide at the cathode. The integration of enzymatic electrosynthesis with EFC architectures can simultaneously result in self-powered enzymatic electrosynthesis with more valuable usage of electrons to produce high-value-added chemicals. In this study, a H2/α-keto acid EFC was developed for the conversion from chemically inert nitrogen gas to chiral amino acids, powered by H2 oxidation. A highly efficient cathodic reaction cascade was first designed and constructed. Powered by an applied voltage, the cathode supplied enough reducing equivalents to support the NH3 production and NADH recycling catalyzed by nitrogenase and diaphorase. The produced NH3 and NADH were reacted in situ with leucine dehydrogenase (LeuDH) to generate l-norleucine with 2-ketohexanoic acid as the NH3 acceptor. A 92% NH3 conversion ratio and 87.1% Faradaic efficiency were achieved. On this basis, a H2-powered fuel cell with hyper-thermostable hydrogenase (SHI) as the anodic catalyst was combined with the cathodic reaction cascade to form the H2/α-keto acid EFC. After 10 h of reaction, the concentration of l-norleucine achieved 0.36 mM with >99% enantiomeric excess and 82% Faradaic efficiency. From the broad substrate scope and the high enzymatic enantioselectivity of LeuDH, the H2/α-keto acid EFC is an energy-efficient alternative to electrochemically produce chiral amino acids for biotechnology applications.
- Cai, Rong,Chen, Hsiaonung,Chen, Hui,Dong, Fangyuan,Minteer, Shelley D.,Prater, Matthew B.
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supporting information
p. 4028 - 4036
(2020/03/11)
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- Artificial Biocatalytic Cascade with Three Enzymes in One Pot for Asymmetric Synthesis of Chiral Unnatural Amino Acids
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Two biocatalytic reactions, transamination catalyzed by transaminases and reductive amination catalyzed by amino acid dehydrogenases, can be used for asymmetric synthesis of optically pure unnatural amino acids. However, although transaminases show a great diversity and broad substrate spectrum, most transaminase reactions are reversible, while amino acid dehydrogenases catalyze reductive amination irreversibly but with strict substrate specificity. Accordingly, herein we developed a tri-enzyme one-pot reaction system to exploit the respective advantages of transaminases and amino acid dehydrogenases, while overcoming the disadvantages of each. In this work, representatives of all four subgroups of transaminases coupled with different amino acid dehydrogenases to produce five l- and four d- unnatural amino acid products, using ammonia and the co-enzyme NAD(P)H, which is regenerated by a robust alcohol dehydrogenase with 2-propanol as cheap cosubstrate. The complete conversion and high enantiopurity (ee > 99 %) of the products, demonstrated it as an ideal alternative for asymmetric synthesis of chiral amino acid compounds.
- Zhou, Haisheng,Meng, Lijun,Yin, Xinjian,Liu, Yayun,Xu, Gang,Wu, Jianping,Wu, Mianbin,Yang, Lirong
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supporting information
p. 6470 - 6477
(2019/11/02)
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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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- 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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p. 3309 - 3314
(2018/10/02)
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- A process for preparing L-tert-leucine method
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The invention discloses a new method for preparing L-tertiary leucine. The new method comprises the following steps: 1, performing asymmetric cyanosilylation reaction on pivalaldehyde, L-phenylglycinol and trimethylsilyl cyanide in the presence of a catalyst A, and carrying out posttreatment to obtain a compound I; 2, hydrolyzing the compound I under acidic condition, and carrying out posttreatment to obtain a compound II; 3, performing catalytic hydrogenation reaction on the compound II to obtain L-tertiary leucine, wherein the catalyst A is magnesium diiodide, magnesium dibromide, magnesium dichloride, magnesium perchlorate or magnesium trifluoromethanesulfonate. The stereochemistry of the L-tertiary leucine can be constructed with high selectivity under solvent-free condition by adopting a magnesium catalyzed three-component one-pot asymmetric cyanosilylation reaction through cheap and easily available organic raw materials.
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Paragraph 0017; 0054; 0055
(2017/01/17)
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- Method for production of L-amino acid
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The present invention has its object to provide a method for producing an L-amino acid comprising reacting a keto acid with an amino acid dehydrogenase and an enzyme having coenzyme regenerating ability to convert to a L-amino acid, wherein a coenzyme is added in two or more portions in the reaction. The method of the present invention enables efficient production of an L-amino acid useful as a synthetic intermediate such as a pharmaceutical intermediate with high optical purity by an enzymatic reductive amination independent of the purity of the keto acid used as a substrate.
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Page/Page column 9
(2016/03/19)
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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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- Resolution of α/β-amino acids by enantioselective penicillin G acylase from Achromobacter sp.
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Penicillin G acylases (PGAs) are enantioselective enzymes catalyzing a hydrolysis of stable amide bond in a broad spectrum of substrates. Among them, derivatives of α- and β-amino acids represent a class of compounds with high application potential. PGAEc from Escherichia coli and PGAA from Achromobacter sp. CCM 4824 were used to catalyze enantioselective hydrolyses of seven selected N-phenylacetylated α/β-amino acid racemates. The PGAA showed higher stereoselectivity for enantiomers of N-PhAc-β-homoleucine, N-PhAc-α-tert-leucine and N-PhAc-β-leucine. To study the mechanism of enantiodiscrimination on molecular level, we have constructed a homology model of PGAA that was used in molecular docking experiments with the same substrates. In-silico experiments successfully reproduced the data from experimental enzymatic resolutions confirming validity of employed modeling protocol. We employed this protocol to evaluate enantiopreference of PGAA towards seven new substrates with application potential. For five of them, high enantioselectivity of PGAA was predicted.
- Grulich, Michal,Brezovsky, Jan,?těpánek, Václav,Palyzová, Andrea,Kyslíková, Eva,Kyslík, Pavel
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p. 240 - 247
(2015/10/28)
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- PROCESS FOR PRODUCING SOLID AMINO ACID
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The problem to be solved by the present invention is to ea lily and efficiently produce an amino acid having 2 to 7 carbon atoms as a high-purity solid without complicated operation, which is useful as a synthetic intermediate for medicines or agrochemicals. The present invention is characterized in comprising a step of precipitating solid amino acid with high purity. In the present invention, the by-produced salt composed of the sulfonic acid and the amine was removed to the mother liquor by reacting an amine with a sulfonic acid salt of amino acid in an aprotic polar solvent, or by reacting a sulfonic acid with an amine salt of amino acid in an aprotic polar solvent. The sulfonic acid salt of amino acid, for example, may be produced by reacting a N-(tert-butoxycarbonyl) amino acid with a sulfonic acid, or by reacting an amino acid tert-butyl ester with a sulfonic acid.
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Paragraph 0067-0069
(2014/12/09)
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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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- 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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p. 391 - 400
(2014/01/23)
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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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- A process for the preparation of enantiomerically pure tert-leucine
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A Process for the Preparation of Enantiomerically Pure tert-Leucine : Enantiomerically pure L-tert-leucine and D-tert-leucine were prepared from (DL)-tert-leucine by diastereomeric salt formation using dibenzoyl-d-tartaric acid as the resolving agent.
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Page/Page column 4
(2012/10/18)
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- PROCESS FOR PRODUCTION OF N-ALKOXYCARBONYL-tert-LEUCINES
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A high quality N-alcoxycarbonyl-tert leucine can be efficiently and stably produced with an easy procedure by reacting tert-leucine with an N-alcoxycarbonylating agent in the presence of water, wherein the use amount of the N-alcoxycarbonylating agent is not less than 0.90 times by mole and not more than 1.00 times by mole relative to the tert-leucine, with maintaining the pH of the reaction mixture in the range of not less than 9 and not more than 13 using a basic agent. In addition, an N-alcoxycarbonyl-tert-leucine can be efficiently extracted from the basic aqueous solution thereof under a mild condition using a water-immiscible solvent by mixing a hydroxide. Furthermore, the two crystal forms of N-butoxycarbonyl-tert-leucine can be controlled by adjusting the water amount in crystallization step, and the compound can be stably produced in an industrial production.
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Page/Page column 15
(2012/03/26)
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- PROCESS FOR THE PREPARATION OF ENANTIOMERICALLY PURE tert-LEUCINE
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Enantiomerically pure L-tert-leucine and D-tert-leucine were prepared from (DL)-tert-leucine by diastereomeric salt formation using dibenzoyl-d-tartaric acid as the resolving agent.
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Page/Page column 2-3
(2012/10/08)
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- Polymer-supported enantioselective bifunctional catalysts for nitro-Michael addition of ketones and aldehydes
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Introduction of an L-amino acid as a spacer and a urea-forming moiety in a polymer-supported bifunctional urea-primary amine catalyst, based on (1R, 2R)-(+)-1,2-diphenylethylenediamine, significantly improves the catalyst's activity and stereoselectivity in the asymmetric addition of ketones and aldehydes to nitroolefins. Yields and enantioselectivities, unprecedented for immobilized catalysts, were obtained with such challenging donors as acetone, cyclopentanone, and α,α-disubstituted aldehydes, which usually perform inadequately in this reaction (particularly when a secondary-amine-based catalyst is used). Remarkably, though in the examined catalysts the D-amino acids as spacers were significantly inferior to the L isomers, for the chosen configuration of the diamine (match-mismatch pairs) the size of the side chain of the amino acid hardly influenced the enantioselectivity of the catalyst. These results, combined with the reactivity profile of the catalysts with substrates bearing two electron-withdrawing groups and the behavior of the catalysts' analogues based on tertiary (rather than primary) amine, suggest an enamine-involving addition mechanism and a particular ordered C-C bond-forming transition state as being responsible for the catalytic reactions with high enantioselectivity. Copyright
- Tuchman-Shukron, Lital,Miller, Scott J.,Portnoy, Moshe
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p. 2290 - 2296
(2012/03/27)
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- L-SUCCINYLAMINOACYLASE AND PROCESS FOR PRODUCING L-AMINO ACID USING IT
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The present invention provides an L-aminoacylase which is able to produce L-tert-leucine being useful as an intermediate for pharmaceuticals. A protein which is characterized in being represented by any of the following (a) to (d): (a) a protein coded by a gene consisting of a nucleic acid sequence shown in SEQ ID No: 1; (b) a protein consisting of an amino acid sequence shown in SEQ ID No: 2; (c) a protein coded by a polynucleotide which hybridizes under a stringent condition with a nucleic acid sequence which is complementary to the nucleic acid sequence shown in SEQ ID No: 1 and having an L-succinylaminoacylase activity; and (d) a protein which consists of an amino acid sequence where one or several amino acid (s) is/are substituted, deleted, inserted and/or added in the protein consisting of the amino acid sequence shown in SEQ ID No: 2 and has an L-succinylaminoacylase activity.
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- Asymmetric synthesis of l-tert-leucine and l-3-hydroxyadamantylglycine using branched chain aminotransferase
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l-Tert-leucine (Tle) and l-3-hydroxyadamantylglycine of (HAG) are important intermediates for a variety of pharmaceutical classes. They were asymmetrically produced from corresponding keto acids using branched-chain aminotransferase (BCAT) with l-glutamate (Glu) as an amino donor. For the production of l-Tle and l-HAG, BCAT from Enterobacter sp. TL3 (BCATen) and BCAT from Escherichia coli K12 (ilvE, newly named as BCATes) were used, respectively. In our current study, we characterized the basic properties of BCATen and BCATes such as substrate specificity, enantioselectivity, and kinetic parameters. The activities of BCATen and BCATes were inhibited severely by α-ketoglutarate which is a deaminated product of l-Glu. In the presence of 10 mM α-ketoglutarate, both enzymes activities were reduced up to 80%. In order to overcome product inhibition by α-ketoglutarate and the problem of equilibrium of the transamination reaction, coupling reactions were carried out with l-glutamate dehydrogenase (GDH)/formate dehydrogenase (FDH) and AspAT. The coupling reaction dramatically increased the yields of both target compounds. 135 mM of l-Tle (>99% ee) was produced from 150 mM corresponding keto acid in BCATen/GDH/FDH coupling reaction with 90% conversion. In addition, 90.5 mM l-HAG (>99% ee) was produced from 100 mM corresponding keto acid in BCATes/AspAT coupling reaction using recombinant whole-cells.
- Hong, Eun Young,Cha, Minho,Yun, Hyungdon,Kim, Byung-Gee
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body text
p. 228 - 233
(2011/01/03)
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- Scaleable catalytic asymmetric Strecker syntheses of unnatural α-amino acids
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α-Amino acids are the building blocks of proteins and are widely used as components of medicinally active molecules and chiral catalysts. Efficient chemo-enzymatic methods for the synthesis of enantioenriched α-amino acids have been developed, but it is still a challenge to obtain non-natural amino acids. Alkene hydrogenation is broadly useful for the enantioselective catalytic synthesis of many classes of amino acids, but it is not possible to obtain α-amino acids bearing aryl or quaternary alkyl α-substituents using this method. The Strecker synthesisthe reaction of an imine or imine equivalent with hydrogen cyanide, followed by nitrile hydrolysisis an especially versatile chemical method for the synthesis of racemic α-amino acids. Asymmetric Strecker syntheses using stoichiometric amounts of a chiral reagent have been applied successfully on gram-to-kilogram scales, yielding enantiomerically enriched α-amino acids. In principle, Strecker syntheses employing sub-stoichiometric quantities of a chiral reagent could provide a practical alternative to these approaches, but the reported catalytic asymmetric methods have seen limited use on preparative scales (more than a gram). The limited utility of existing catalytic methods may be due to several important factors, including the relatively complex and precious nature of the catalysts and the requisite use of hazardous cyanide sources. Here we report a new catalytic asymmetric method for the syntheses of highly enantiomerically enriched non-natural amino acids using a simple chiral amido-thiourea catalyst to control the key hydrocyanation step. This catalyst is robust, without sensitive functional groups, so it is compatible with aqueous cyanide salts, which are safer and easier to handle than other cyanide sources; this makes the method adaptable to large-scale synthesis. We have used this new method to obtain enantiopure amino acids that are not readily prepared by enzymatic methods or by chemical hydrogenation.
- Zuend, Stephan J.,Coughlin, Matthew P.,Lalonde, Mathieu P.,Jacobsen, Eric N.
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scheme or table
p. 968 - 970
(2010/06/16)
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- Enzymatic approach to both enantiomers of N-Boc hydrophobic amino acids
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Protease catalysed hydrolysis of N-Boc-amino acid esters allows us to obtain N-Boc l-acids and d-esters of amino butanoic acid, nor-leucine, nor-valine, leucine and t-leucine in excellent ee. The reaction occurs in short reaction times and high concentrations. When a biphasic system (buffer-MTBE) is employed, a strong solvent effect is observed. This method could be of significance for the preparation of d-t-leucine, for which a practical method is currently unavailable.
- Agosta, Eleonora,Caligiuri, Antonio,D'Arrigo, Paola,Servi, Stefano,Tessaro, Davide,Canevotti, Renato
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p. 1995 - 1999
(2007/10/03)
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- PROCESS FOR PRODUCING TERT-LEUCINE
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The present invention provides a method of producing tert-leucine, which includes reacting tert-leucine with substituted benzenesulfonic acid represented by the following formula (1) wherein R1 and R2 are each independently a hydrogen atom, an alkyl group or a halogen atom, except a compound wherein R1 and R2 are both hydrogen atoms, in a solvent, to give tert-leucine·substituted benzenesulfonate represented by the following formula (2) wherein R1 and R2 are the same substituents as above, separating the salt by crystallization and then dissociating the salt. According to the present invention, a production method of tert-leucine is provided, which includes forming a tert-leucine salt hardly soluble in a solvent and crystallization thereof to efficiently recover tert-leucine from the solvent.
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Page/Page column 6
(2008/06/13)
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- Process for producing optically active alpha-amino acid and optically active alpha-amino acid amine
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The present invention provides a process for efficiently producing an optically active α-amino acid and an optically active α-amino acid amide. After contacting with cells or processed cells thereof having an ability to asymmetrically hydrolyse, a water solvent is substituted with at least one solvent selected from the group consisting of linear, branched, or cyclic alcohol having 3 or more carbon atoms and the optically active α-amino acid is preferentially precipitated from the alcohol solution. The addition of basic compounds, particularly potassium compounds to the alcohol solution containing the optically active α-amino acid amide, which is obtained after the separation of the optically active α-amino acid, enables the purification of the amide without the inclusion of amino acid into amino acid amide. Thus, the amide is subjected to the step of racemization and then recycled.
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- Orthoacylimines: A new class of chiral auxiliaries for nucleophilic addition of organolithium reagents to imines
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A new class of orthoacylimine-derived chiral auxiliaries has been synthesized and tested in the nucleophilic addition of organolithium reagents to imines. The precursors can be prepared by an aza-Wittig reaction between the corresponding orthoacyl azide and a variety of aldehydes in the presence of trialkylphosphines. The nucleophilic addition of organolithium reagents led to the addition products in good yields and with good to excellent diastereoselectivities (from 85:15 to 99:1). The chiral, nonracemic secondary amines could be readily obtained under mild hydrolytic condition. Furthermore, the chiral auxiliary can be recovered in quantitative yield and reconverted to the starting orthoacyl azide precursor. This method was applied to the synthesis of (S)-t-leucine.
- Boezio, Alessandro A.,Solberghe, Geoffrey,Lauzon, Caroline,Charette, Andre B.
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p. 3241 - 3245
(2007/10/03)
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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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p. 533 - 538
(2013/09/06)
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- Method for preparing tert-leucine and analogues thereof in enantiomeric form and intermediates therein
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Azlactone (3), or the opposite enantiomer thereof, undergoes biotransformation, using suitable enzymatic activity, in the presence of a compound YH to form a N-acyl-amino acid (2), wherein R1, R2 and R3 are each not hydrogen and are independently selected from groups containing up to 20 carbon atoms, optionally with any combination of R1, R2 and R3 being joined together to form at least one ring, X is selected from groups containing up to 20 carbon atoms, and Y is selected from the group consisting of —OH, -Oalkyl and -Nalkyl. Amino acid (1), or the opposite enantiomer thereof, can be prepared in high enantiomeric excess from N-acyl amino acid (2), by converting Y to OH.
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- Asymmetric strecker synthesis of α-amino acids via a crystallization-induced asymmetric transformation using (R)-phenylglycine amide as chiral auxiliary
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Matrix presented Diastereoselective Strecker reactions based on (R)-phenylglycine amide as chiral auxiliary are reported. The Strecker reaction is accompanied by an in situ crystallization-induced asymmetric transformation, whereby one diastereomer selectively precipitates and can be isolated in 76-93% yield and dr > 99/1. The diastereomerically pure α-amino nitrile obtained from pivaldehyde (R1 = t-Bu, R2 = H) was converted in three steps to (S)-tert-leucine in 73% yield and >98% ee.
- Boesten, Wilhelmus H. J.,Seerden, Jean-Paul G.,De Lange, Ben,Dielemans, Hubertus J. A.,Elsenberg, Henk L. M.,Kaptein, Bernard,Moody, Harold M.,Kellogg, Richard M.,Broxterman, Quirinus B.
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p. 1121 - 1124
(2007/10/03)
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- Thermodynamics of reactions catalysed by branched-chain-amino-acid transaminase
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Apparent equilibrium constants and calorimetric enthalpies of reaction have been measured for reactions catalysed by branched-chain-amino-acid transaminase. The following biochemical reactions have been studied at the temperature 298.15 K and in the pH ra
- Tewari, Yadu B.,Goldberg, Robert N.,Rozzell, J. David
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p. 1381 - 1398
(2007/10/03)
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- Optically active iminocarboxylic acid derivatives
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The compounds of the formula STR1 wherein R is OH, NH2, lower-alkyl-NH or phenyl-lower alkyl-NH are presented. These compounds can be catalytically hydrogenated to the corresponding α-aminocarboxylic acid derivatives which are intermediates in the synthesis of therapeutic pseudopeptides.
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- Enantioselective synthesis of α-amino acids from nitroalkenes
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The products of the conjugate addition of (R)-4-phenyl-2-oxazolidinone on monosubstituted nitroalkenes, were converted into D-α-amino acids of high enantiomeric purity.
- Sabelle, Stephane,Lucet, Denis,Le Gall, Thierry,Mioskowski, Charles
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p. 2111 - 2114
(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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- Synthesis of homochiralL-(S)-tert-leucine via a lipase catalysed dynamic resolution process
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Treatment of (±)-2-phenyl-4-tert-butyloxazolin-5(4H)-one 8 with Lipozyme (Mucor miehei) in toluene containing n-butanol and a catalytic amount of triethylamine resulted in a 94% yield of (S)-N-benzoyltert-leucine butyl ester 9 (99.5% e.e.). Subsequent two step hydrolysis (Alcalase followed by 6N HCl, reflux) yielded homochiral L-(S)-tert-leucine 1.
- Turner, Nicholas J.,Winterman, James R.,McCague, Raymond,Parratt, Julian S.,Taylor, Stephen J. C.
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p. 1113 - 1116
(2007/10/02)
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- AMINOACID DERIVATIVES INHIBITING RENIN
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New amino acid derivatives of the formulaR. sup.1--Z--NR 2--CHR 3--CR 4--(CHR 5) a--CO--E--Q--Ywherein R 1 to R 5, a, Z, E, Q and Y have the meanings defined herein, and salts thereof inhibit the activity of human plasma renin.
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- Optically active ester derivatives, preparation process thereof, liquid crystal materials and a light switching element
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Disclosed are herein optically active ester derivatives represented by the formula (I): STR1 (wherein R1 represents an alkyl group having 3 to 20 carbon atoms; R2 represents an optically active alkyl or alkoxyalkyl group having 3 to 15 carbon atoms optionally substituted by halogen atoms; Y represents --O--, --COO-- or --OCO--; X represents --COO-- or --OCO--; l represents a number of 1 or 2; k and m each represents a number of 0 or 1; n represents a number of 1 to 6), preparation processes therefor, liquid crystal materials containing such ester derivatives as active ingredient, and a light switching element using said liquid crystal materials as liquid crystal element.
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- An Efficient and Practical Synthesis of L-α-Amino Acids Using (R)-Phenylglycinol as a Chiral Auxiliary
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L-α-Amino acids including L-α-arylglycines were conveniently and stereoselectively synthesized via the α-amino carbonitriles given by the Strecker reaction of (R)-2-amino-2-phenylethanol with aldehydes and hydrogen cyanide.The stereoselectivity of these α-amino carbonitriles was thermodynamically controlled.
- Inaba, Takashi,Kozono, Ichiro,Fujita, Makoto,Ogura, Katsuyuki
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p. 2359 - 2365
(2007/10/02)
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- Stereoselective synthesis of L-amino acids via Strecker and Ugi reactions on carbohydrate templates
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L-Amino acid derivatives are stereoselectively synthesized in high yield using 2,3,4-tri-O-pivaloyl-α-D-arabinopyranosylamine or 2,3,4-tri-O-pivaloyl-β-L-fucopyranosylamine as the chiral auxiliary in Strecker and Ugi reactions.
- Kunz,Pfrengle,Ruck,Sager
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p. 1039 - 1042
(2007/10/02)
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- Kinetic Resolution of Unnatural and Rarely Occuring Amino Acids: Enantioselective Hydrolysis of N-Acyl Amino Acids Catalyzed by Acylase I
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Acylase I (aminoacylase; N-acylamino-acid amidohydrolase, EC 3.5.1.14, from porcine kidney and the fungus Aspergillus) is broadly applicable enzymatic catalyst for the kinetic resolution of unnatural and rarely occuring α-amino acids.Its enantioselectivity for the hydrolysis of N-acyl L-α-amino acids is nearly absolute, yet it accepts substrates having a wide range of structure and functionality.This paper reports the initial rates of enzyme-catalyzed hydrolysis of over 50 N-acyl amino acids and analogues, the stabilities of the enzymes in aqueous and aqueous/organic solutions, and the effects of different acyl groups and metal ions on the rates of enzymatic hydrolysis.Eleven α-amino and α-methyl α-amino acids were resolved on a 2-29-g scale.Crude L- and D-amino acid products had generally >90percent ee.The utility of resolved amino acids as chiral synthons was illustrated by the preparation of (R)- and (S)-1-butene oxide and the diastereoselective (cis:trans, 7-8:1) iodolactonization of three 2-amino-4-alkenoic acid derivatives.
- Chenault, H. Keith,Dahmer, Juergen,Whitesides, George M.
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p. 6354 - 6364
(2007/10/02)
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- CARBOHYDRATES AS CHIRAL TEMPLATES: DIASTEREOSELECTIVE UGI SYNTHESIS OF (S)-AMINO ACIDS USING O-ACYLATED D-ARABINOPYRANOSYLAMINE AS THE AUXILIARY
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Enantiomerically pure (S)-amino acids are synthesized via a highly diastereoselective Ugi reaction using 2,3,4-tri-O-pivaloyl-α-D-arabinopyranosylamine as the chiral template.
- Kunz, Horst,Pfrengle, Waldemar,Sager, Wilfried
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p. 4109 - 4110
(2007/10/02)
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