- One-step Synthesis of α-Keto Acids from Racemic Amino Acids by A Versatile Immobilized Multienzyme Cell-free System
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The elevated value of α-keto acids has pushed scientists to explore more efficient and less expensive alternatives for their synthesis. In this work, an immobilized tri-enzyme system that produced α-keto acids in “one-pot” from l- or racemic mixtures of diverse amino acids was presented. The system combined a broad-spectrum amino acid racemase (BsrV), a d-amino acid oxidase (DAAO) and catalase (CAT). BsrV racemized l-amino acids into their d-enantiomers, DAAO catalyzed the stereospecific oxidative deamination of the d-amino acids into their corresponding α-keto acids, ammonium ion, and H2O2. Finally, CAT converted the inactivating H2O2 into H2O and O2, which can be reused by the oxidase reaction. BsrV thermal stability was improved 3,300-fold by immobilizing the enzyme on glyoxyl-activated agarose beads. DAAO and CAT were co-immobilized on agarose beads functionalized with glutaraldehyde groups for enhancing their stabilities and eliminating H2O2 in a much more effective way. To show the versatility of this system, racemic mixtures of amino acids were converted in their corresponding α-keto acids. The coupling of the three immobilized enzymes permitted conversions of approximately 99 % through a dynamic kinetic resolution process. This system conserved 100 % of its initial effectiveness after 8 reaction cycles. Collectively, our innovative tri-enzyme system for the synthesis of α-keto acids opens the door for a cheapening in the production of many pharmaceutical and cosmetics.
- Orrego, Alejandro H.,López-Gallego, Fernando,Espaillat, Akbar,Cava, Felipe,Guisan, José M.,Rocha-Martin, Javier
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- Structural and functional evolution of isopropylmalate dehydrogenases in the leucine and glucosinolate pathways of Arabidopsis thaliana
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The methionine chain-elongation pathway is required for aliphatic glucosinolate biosynthesis in plants and evolved from leucine biosynthesis. In Arabidopsis thaliana, three 3-isopropylmalate dehydrogenases (AtIPMDHs) play key roles in methionine chain-elongation for the synthesis of aliphatic glucosinolates (e.g. AtIPMDH1) and leucine (e.g. AtIPMDH2 and AtIPMDH3). Here we elucidate the molecular basis underlying the metabolic specialization of these enzymes. The 2.25 A° resolution crystal structure of AtIPMDH2 was solved to provide the first detailed molecular architecture of a plant IPMDH. Modeling of 3-isopropylmalate binding in the AtIPMDH2 active site and sequence comparisons of prokaryotic and eukaryotic IPMDH suggest that substitution of one active site residue may lead to altered substrate specificity and metabolic function. Sitedirected mutagenesis of Phe-137 to a leucine in AtIPMDH1 (AtIPMDH1-F137L) reduced activity toward 3-(2′-methylthio)-ethylmalate by 200-fold, but enhanced catalytic efficiency with 3-isopropylmalate to levels observed with AtIPMDH2 and AtIPMDH3. Conversely, the AtIPMDH2-L134F and AtIPMDH3-L133F mutants enhanced catalytic efficiency with 3-(2′-methylthio)ethylmalate ~100-fold and reduced activity for 3-isopropylmalate. Furthermore, the altered in vivo glucosinolate profile of an Arabidopsis ipmdh1 T-DNA knock-out mutant could be restored to wild-type levels by constructs expressing AtIPMDH1, AtIPMDH2-L134F, or AtIPMDH3-L133F, but not by AtIPMDH1-F137L. These results indicate that a single amino acid substitution results in functional divergence of IPMDH in planta to affect substrate specificity and contributes to the evolution of specialized glucosinolate biosynthesis from the ancestral leucine pathway.
- He, Yan,Galant, Ashley,Pang, Qiuying,Strul, Johanna M.,Balogun, Sherifat F.,Jez, Joseph M.,Chen, Sixue
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- Stereospecificity of the Hydride Transfer Reaction Catalyzed by Isopropylmalate Dehydrogenase of Thermophilic Bacteria Thermus thermophilius
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Nuclear magnetic resonance studied on the NAD-dependent reaction catalyzed by isopropylmalate dehydrogenase from T. thermophilius HB8 revealed that pro R specific (A specific) hydride transfer from the substrate to the nicotinamide ring is involved during the said oxido-reduction.
- Yamada, Tohru,Kakinuma, Katsumi,Endo, Toyoshige,Oshima, Tairo
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- Coenzyme activity of NAD analogs for 3-isopropylmalate dehydrogenase from Thermus thermophilus HB8
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In order to elucidate the enzyme-substrate-cofactor interaction in 3-isopropylmalate dehydrogenase, the coenzyme activity of NAD analogs which have a 3-substituted pyridine ring was examined. Analogs 3-5 showed diminished kcat values compared with those of NAD+, whereas thiocarboxamide 2 was almost as equally active as NAD+. This suggests that the NH2 functionality of NAD+ is more important for the catalysis of IPMDH than a carbonyl group.
- Chiba, Akira,Eguchi, Tadashi,Oshima, Tairo,Kakinuma, Katsumi
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- Overproduction and substrate specificity of 3-isopropylmalate dehydrogenase from Thiobacillus ferrooxidans.
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We constructed an overexpression system in Escherichia coli of the leuB gene coding for 3-isopropylmalate dehydrogenase in Thiobacillus ferrooxidans. E. coli harboring the plasmid we constructed, pKK leuB1, produced 17-fold the enzyme protein of the expression system previously used for purification. The substrate specificity of the enzyme was analyzed with synthetic (2R, 3S)-3-alkylmalates. The 3-isopropylmalate dehydrogenase of Thiobacillus ferrooxidans had broad specificity toward the alkylmalates.
- Matsunami,Kawaguchi,Inagaki,Eguchi,Kakinuma,Tanaka
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- Palladium-Catalyzed β-Arylation of α-Keto Esters
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A catalyst system derived from commercially available Pd2(dba)3 and PtBu3 has been applied to the coupling of α-keto ester enolates and aryl bromides. The reaction provides access to an array of β-stereogenic α-keto esters. When the air-stable ligand precursor PtBu3·HBF4 is employed, the reaction can be carried out without use of a glovebox. The derived products are of broad interest given the prevalence of the α-keto acid substructure in biologically important molecules.
- Zavesky, Blane P.,Bartlett, Samuel L.,Johnson, Jeffrey S.
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- Scalable and Selective β-Hydroxy-α-Amino Acid Synthesis Catalyzed by Promiscuous l-Threonine Transaldolase ObiH
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Enzymes from secondary metabolic pathways possess broad potential for the selective synthesis of complex bioactive molecules. However, the practical application of these enzymes for organic synthesis is dependent on the development of efficient, economical, operationally simple, and well-characterized systems for preparative scale reactions. We sought to bridge this knowledge gap for the selective biocatalytic synthesis of β-hydroxy-α-amino acids, which are important synthetic building blocks. To achieve this goal, we demonstrated the ability of ObiH, an l-threonine transaldolase, to achieve selective milligram-scale synthesis of a diverse array of non-standard amino acids (nsAAs) using a scalable whole cell platform. We show how the initial selectivity of the catalyst is high and how the diastereomeric ratio of products decreases at high conversion due to product re-entry into the catalytic cycle. ObiH-catalyzed reactions with a variety of aromatic, aliphatic and heterocyclic aldehydes selectively generated a panel of β-hydroxy-α-amino acids possessing broad functional-group diversity. Furthermore, we demonstrated that ObiH-generated β-hydroxy-α-amino acids could be modified through additional transformations to access important motifs, such as β-chloro-α-amino acids and substituted α-keto acids.
- Buller, Andrew R.,Doyon, Tyler J.,Grieger, Abbigail M.,Kim, Maeve,Kumar, Prasanth,Madigan, Cormac,Stitgen, Abigail,Thein, Sierra,Willoughby, Patrick H.
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- Chemoenzymatic Production of Enantiocomplementary 2-Substituted 3-Hydroxycarboxylic Acids from l-α-Amino Acids
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A two-enzyme cascade reaction plus in situ oxidative decarboxylation for the transformation of readily available canonical and non-canonical l-α-amino acids into 2-substituted 3-hydroxycarboxylic acid derivatives is described. The biocatalytic cascade consisted of an oxidative deamination of l-α-amino acids by an l-α-amino acid deaminase from Cosenzaea myxofaciens, rendering 2-oxoacid intermediates, with an ensuing aldol addition reaction to formaldehyde, catalyzed by metal-dependent (R)- or (S)-selective carboligases namely 2-oxo-3-deoxy-l-rhamnonate aldolase (YfaU) and ketopantoate hydroxymethyltransferase (KPHMT), respectively, furnishing 3-substituted 4-hydroxy-2-oxoacids. The overall substrate conversion was optimized by balancing biocatalyst loading and amino acid and formaldehyde concentrations, yielding 36–98% aldol adduct formation and 91–98% ee for each enantiomer. Subsequent in situ follow-up chemistry via hydrogen peroxide-driven oxidative decarboxylation afforded the corresponding 2-substituted 3-hydroxycarboxylic acid derivatives. (Figure presented.).
- Pickl, Mathias,Marín-Valls, Roser,Joglar, Jesús,Bujons, Jordi,Clapés, Pere
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p. 2866 - 2876
(2021/04/14)
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- Total Synthesis of the Natural Herbicide MBH-001 and Analogues
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The first total synthesis of the natural herbicide MBH-001 (1) is reported. Structurally it is a 2-methyloxazol-5(2H)-one with a (1-hydroxyethyl) substituent at the 2-position. By relying on cyclic nitrones, a flexible route to MBH-001 and relevant analogues was developed. Key steps include the reaction of a 2-hydroxyimino ester with an aldehyde to form a 5-oxo-2,5-dihydrooxazole 3-oxide. In an aldol-type reaction, the anion of these cyclic nitrones reacted with an aldehyde at the 2-position. A final reduction of the nitrone to the corresponding imine using zinc led to the target compounds. The cyclic nitrones are also accessible by reacting an α-keto acid with an oxime. These two versatile synthetic routes enabled us to prepare the first MBH-001 analogues for structure activity relationship analysis of the herbicidal efficacy. Thus, furthering our aim of developing new herbicides to tackle the ever-growing problem of weed resistance.
- Barber, David M.,D?ller, Uwe,Dietrich, Hansj?rg,Hoffmann, Michael G.,Kocakaya, Tamer,Kuhn, Birgit,Maier, Martin E.,Morkunas, Marius,Schmutzler, Dirk,Schnatterer, Stefan
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p. 2271 - 2290
(2020/04/23)
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- Exploration of Transaminase Diversity for the Oxidative Conversion of Natural Amino Acids into 2-Ketoacids and High-Value Chemicals
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The use of 2-ketoacids is very common in feeds, food additives, and pharmaceuticals, and 2-ketoacids are valuable precursors for a plethora of chemically diverse compounds. Biocatalytic synthesis of 2-ketoacids starting from l-amino acids would be highly desirable because the substrates are readily available from biomass feedstock. Here, we report bioinformatic exploration of a series of aminotransferases (ATs) to achieve the desired conversion. Thermodynamic control was achieved by coupling an l-glutamate oxidation reaction in the cascade for the recycling of the amine acceptor. These enzymes were able to convert a majority of proteinogenic amino acids into the corresponding 2-ketoacids with high conversion (up to 99percent) and atom-efficiency. Furthermore, this enzyme cascade was extendable, and one-pot two-step processes were established for the synthesis of d-amino acids and N-methylated amino acids, achieving great overall conversion (up to 99percent) and high ee values (>99percent). These developed enzymatic methodologies offer convenient routes for utilizing amino acids as synthetic reagents.
- Chen, Yanchun,Cui, Xuexian,Cui, Yinglu,Li, Chuijian,Li, Ruifeng,Li, Tao,Sun, Jinyuan,Wu, Bian,Zhu, Tong
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p. 7950 - 7957
(2020/08/21)
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- Production of α-Ketoisocaproate and α-Keto-β-Methylvalerate by Engineered L-Amino Acid Deaminase
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This study aimed to develop an efficient enzymatic strategy for industrial production of α-ketoisocaproate (α-KIC) and α-keto-β-methylvalerate (α-KMV) from L-leucine and L-isoleucine, respectively. L-amino acid deaminase from Proteus mirabilis (PmLAAD) was heterologously expressed in E. coli BL21(DE3) and modified to increase its catalytic efficiency by engineering the PmLAAD substrate-binding cavity and entrance tunnel. Four essential residues (Q92, M440, T436, and W438) were identified from structural analysis and molecular dynamics simulations. Residue Q92 was mutated to alanine, and the volume of the binding cavity, enzyme activity, and the kcat/Km value of mutant PmLAAD Q92A increased to 994.2 ?3, 191.36 U mg?1, and 1.23 mM?1 min?1, respectively; consequently, the titer and conversion rate of α-KIC from L-leucine were 107.1 g L?1 and 98.1 %, respectively. For mutant PmLAADT436/W438A, the entrance tunnel, enzyme activity, and the kcat/Km value increased to 1.71 ?, 170.12 U mg?1, and 0.70 mM?1 min?1, respectively; consequently, the titer and conversion rate of α-KMV from L-isoleucine were 98.9 g L?1 and 99.7 %, respectively. Therefore, augmentation of the substrate-binding cavity and entrance tunnel of PmLAAD can facilitate efficient industrial synthesis of α-KIC and α-KMV.
- Yuan, Yuxiang,Song, Wei,Liu, Jia,Chen, Xiulai,Luo, Qiuling,Liu, Liming
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p. 2464 - 2472
(2019/05/10)
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- The pseudoalteromonas luteoviolacea L-amino acid oxidase with antimicrobial activity is a flavoenzyme
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The marine environment is a rich source of antimicrobial compounds with promising pharmaceutical and biotechnological applications. The Pseudoalteromonas genus harbors one of the highest proportions of bacterial species producing antimicrobial molecules. For decades, the presence of proteins with L-amino acid oxidase (LAAO) and antimicrobial activity in Pseudoalteromonas luteoviolacea has been known. Here, we present for the first time the identification, cloning, characterization and phylogenetic analysis of Pl-LAAO, the enzyme responsible for both LAAO and antimicrobial activity in P. luteoviolacea strain CPMOR-2. Pl-LAAO is a flavoprotein of a broad substrate range, in which the hydrogen peroxide generated in the LAAO reaction is responsible for the antimicrobial activity. So far, no protein with a sequence similarity to Pl-LAAO has been cloned or characterized, with this being the first report on a flavin adenine dinucleotide (FAD)-containing LAAO with antimicrobial activity from a marine microorganism. Our results revealed that 20.4% of the sequenced Pseudoalteromonas strains (specifically, 66.6% of P. luteoviolacea strains) contain Pl-laao similar genes, which constitutes a well-defined phylogenetic group. In summary, this work provides insights into the biological significance of antimicrobial LAAOs in the Pseudoalteromonas genus and shows an effective approach for the detection of novel LAAOs, whose study may be useful for biotechnological applications.
- Andreo-Vidal, Andrés,Sanchez-Amat, Antonio,Campillo-Brocal, Jonatan C.
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- Palladium-catalyzed decarboxylative, decarbonylative and dehydrogenative C(sp2)-H acylation at room temperature
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Over the past few decades, an impressive array of C-H activation methodology has been developed for organic synthesis. However, due to the inherent inertness of the C-H bonds (e.g. ~110 kcal mol-1 for the cleavage of C(aryl)-H bonds) harsh reaction conditions have been realized to overcome high energetic transition states resulting in a limited substrate scope and functional group tolerance. Therefore, the development of mild C-H functionalization protocols is in high demand to exploit the full potential of the C-H activation strategy in the synthesis of a complex molecular framework. Although, electron-rich substrates undergo electrophilic metalation under relatively mild conditions, electron-deficient substrates proceed through a rate-limiting C-H insertion under forcing conditions at high temperature. In addition, a stoichiometric amount of toxic silver salt is frequently used in palladium catalysis to facilitate the C-H activation process which is not acceptable from the environmental and industrial standpoint. We report herein, a Pd(ii)-catalyzed decarboxylative C-H acylation of 2-arylpyridines with α-ketocarboxylic acids under mild conditions. The present protocol does not require stoichiometric silver(i) salts as additives and proceeds smoothly at ambient temperature. A novel decarbonylative C-H acylation reaction has also been accomplished using aryl glyoxals as acyl surrogates. Finally, a practical C-H acylation via a dehydrogenative pathway has been demonstrated using commercially available benzaldehydes and aqueous hydroperoxides. We also disclose that acetonitrile solvent is optimal for the acylation reaction at room temperature and has a prominent role in the reaction outcome. Control experiments suggest that the acylation reaction via decarboxylative, decarbonylative and dehydrogenative proceeds through a radical pathway. Thus we disclose a practical protocol for the sp2 C-H acylation reaction.
- Hossian, Asik,Manna, Manash Kumar,Manna, Kartic,Jana, Ranjan
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supporting information
p. 6592 - 6603
(2017/08/16)
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- Preparation method of alpha-ketoleucine calcium dihydrate or alpha-ketophenylalanine calcium monohydrate
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The invention discloses a preparation method of alpha-ketoleucine calcium dihydrate or alpha-ketophenylalanine calcium monohydrate. In an existing method, the quality of a final product is low, and the content of related substances is high. The method comprises the steps that isobutylidene hydantoin or benzal hydantoin is taken as a raw material and generates a pipeline continuous flow ring-opening reaction with an alcohol-water solution of alkali, and then corresponding alpha-ketoacid salt is obtained, wherein the pipeline continuous flow ring-opening reaction is a continuous ring-opening reaction conducted in a pipeline reactor; the obtained alpha-ketoacid salt is firstly extracted with a hydrocarbon solvent or a halohydrocarbon solvent or a ketone solvent or an ether solvent for impurity removal and then acidized to obtain a corresponding crude alpha-ketonic acid product, the crude alpha-ketonic acid product is extracted with the hydrocarbon solvent or the halohydrocarbon solvent or the ketone solvent or the ether solvent for impurity removal, and then a fine alpha-ketonic acid product is obtained; the obtained alpha-ketonic acid is prepared into calcium salt, crystallization is conducted, and the alpha-ketoacid calcium hydrate is obtained. Accordingly, the high-quality and high-purity alpha-ketoleucine calcium dihydrate and alpha-ketophenylalanine calcium monohydrate are obtained.
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Paragraph 0061; 0062; 0063; 0064
(2017/06/02)
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- Bio-inspired enantioselective full transamination using readily available cyclodextrin
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The mimics of vitamin B6-dependent enzymes that catalyzed an enantioselective full transamination in the pure aqueous phase have been realized for the first time through the establishment of a new “pyridoxal 5′-phosphate (PLP) catalyzed non-covalent cyclodextrin (CD)-keto acid inclusion complexes” system, and various optically active amino acids have been obtained.
- Zhang, Shiqi,Li, Guangxun,Liu, Hongxin,Wang, Yingwei,Cao, Yuan,Zhao, Gang,Tang, Zhuo
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p. 4203 - 4208
(2017/02/05)
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- Recombinant expression and characterization of a l-amino acid oxidase from the fungus Rhizoctonia solani
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l-Amino acid oxidases (L-AAOs) catalyze the oxidative deamination of l-amino acids to the corresponding α-keto acids, ammonia, and hydrogen peroxide. l-AAOs are homodimeric enzymes with FAD as a non-covalently bound cofactor. They are of potential interest for biotechnological applications. However, heterologous expression has not succeeded in producing large quantities of active recombinant l-AAOs with a broad substrate spectrum so far. Here, we report the heterologous expression of an active l-AAO from the fungus Rhizoctonia solani in Escherichia coli as a fusion protein with maltose-binding protein (MBP) as a solubility tag. After purification, it was possible to remove the MBP-tag proteolytically without influencing the enzyme activity. MBP-rsLAAO1 and 9His-rsLAAO1 converted basic and large hydrophobic l-amino acids as well as methyl esters of these l-amino acids. The progress of the conversion of l-phenylalanine and l-leucine into the corresponding α-keto acids was determined by HPLC and 1H-NMR analysis of reaction mixtures, respectively. Enzymatic activity was stimulated 50–100-fold by SDS treatment. Km values ranging from 0.9–10?mM and vmax values from 3 to 10?U?mg?1 were determined after SDS activation of 9His-rsLAAO1 for the best substrates. The enzyme displayed a broad pH optimum between pH 7.0 and 9.5. In summary, a successful overexpression of recombinant l-AAO in E. coli was established that results in a promising enzymatic activity and a broad substrate spectrum for biotechnological application.
- Hahn, Katharina,Neumeister, Katrin,Mix, Andreas,Kottke, Tilman,Gr?ger, Harald,Fischer von Mollard, Gabriele
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p. 2853 - 2864
(2017/03/22)
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- Deracemization and Stereoinversion of α-Amino Acids by l-Amino Acid Deaminase
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Enantiomerically pure α-amino acids are compounds of primary interest for the fine chemical, pharmaceutical, and agrochemical sectors. Amino acid oxidases are used for resolving d,l-amino acids in biocatalysis. We recently demonstrated that l-amino acid deaminase from Proteus myxofaciens (PmaLAAD) shows peculiar features for biotechnological applications, such as a high production level as soluble protein in Escherichia coli and a stable binding with the flavin cofactor. Since l-amino acid deaminases are membrane-bound enzymes, previous applications were mainly based on the use of cell-based methods. Now, taking advantage of the broad substrate specificity of PmaLAAD, a number of natural and synthetic l-amino acids were fully converted by the purified enzyme into the corresponding α-keto acids: the fastest conversion was obtained for 4-nitrophenylalanine. Analogously, starting from racemic solutions, the full resolution (ee >99%) was also achieved. Notably, d,l-1-naphthylalanine was resolved either into the d- or the l-enantiomer by using PmaLAAD or the d-amino acid oxidase variant having a glycine at position 213, respectively, and was fully deracemized when the two enzymes were used jointly. Moreover, the complete stereoinversion of l-4-nitrophenylalanine was achieved using PmaLAAD and a small molar excess of borane tert-butylamine complex. Taken together, recombinant PmaLAAD represents an l-specific amino acid deaminase suitable for producing the pure enantiomers of several natural and synthetic amino acids or the corresponding keto acids, compounds of biotechnological or pharmaceutical relevance. (Figure presented.).
- Rosini, Elena,Melis, Roberta,Molla, Gianluca,Tessaro, Davide,Pollegioni, Loredano
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p. 3773 - 3781
(2017/11/13)
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- Chemoselective conversion from α-hydroxy acids to α-keto acids enabled by nitroxyl-radical-catalyzed aerobic oxidation
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The chemoselective oxidation of α-hydroxy acids to α-keto acids catalyzed by 2-azaadamantane N-oxyl (AZADO), a nitroxyl radical catalyst, is described. Although α-keto acids are labile and can easily release CO2 under oxidation conditions, the use of molecular oxygen as a cooxidant enables the desired chemoselective oxidation.
- Furukawa, Keisuke,Inada, Haruki,Shibuya, Masatoshi,Yamamoto, Yoshihiko
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supporting information
p. 4230 - 4233
(2016/09/09)
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- Biocontrolled formal inversion or retention of L -α-amino acids to enantiopure (R)- or (S)-hydroxyacids
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Natural L-α-amino acids and L-norleucine were transformed to the corresponding α-hydroxy acids by formal biocatalytic inversion or retention of absolute configuration. The one-pot transformation was achieved by a concurrent oxidation reduction cascade in aqueous media. A representative panel of enantiopure (R)- and (S)-2-hydroxy acids possessing aliphatic, aromatic and heteroaromatic moieties were isolated in high yield (67-85 %) and enantiopure form (>99 % ee) without requiring chromatographic purification.
- Busto, Eduardo,Grischek, Barbara,Kroutil, Wolfgang,Richter, Nina
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supporting information
p. 11225 - 11228,4
(2015/01/07)
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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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- Substrate profile of an ω-transaminase from Burkholderia vietnamiensis and its potential for the production of optically pure amines and unnatural amino acids
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A new (S)-enantioselective ω-transaminase (ω-TA) gene from Burkholderia vietnamiensis G4 was functionally expressed in Escherichia coli BL21 (DE3), and the purified recombinant N-terminal His-tagged ω-TA (HBV-ω-TA) had a dimeric structure with optimum pH and temperature of 8.4 and 40 C, respectively. The enzyme showed higher activities toward aromatic amines than aliphatic amines and (S)-1-methylbenzylamine ((S)-α-MBA) was the most active amino donor. For amino acceptor, keto acids, keto esters and aldehydes were more reactive than ketones with pyruvate ethyl ester being most active. Several chiral amines and unnatural amino acids or esters were synthesized using HBV-ω-TA as the catalyst and isopropylamine or (S)-α-MBA as amino donor. Notably, HBV-ω-TA catalyzed the amino transfer to β-keto esters to give optically pure β-amino acid esters. In addition, glyoxylate was used as amino acceptor for the first time in the kinetic resolution of racemic amines and optically pure amines, such as (R)-1-methylbenzylamine, (R)-1-phenylpropylamine, (R)-2-amino-4-phenylbutane and (R)-1-aminotetraline, were obtained.
- Jiang, Jinju,Chen, Xi,Feng, Jinhui,Wu, Qiaqing,Zhu, Dunming
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- Synthesis of the reported structure of piperazirum using a nitro-Mannich reaction as the key stereochemical determining step
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Piperazirum, isolated from Arum palaestinum Boiss, was originally assigned as r-3,c-5-diisobutyl-c-6-isopropylpiperazin-2-one. The reported structure was synthesised diastereoselectively using a key nitro-Mannich reaction to set up the C5/C6 relative stereochemistry. The structure was unambiguously assigned by single crystal X-ray diffraction but the spectroscopic data did not match those reported for the natural product. The structure of the natural product must therefore be revised.
- Anderson, James C.,Kalogirou, Andreas S.,Porter, Michael J.,Tizzard, Graham J.
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p. 1737 - 1744
(2013/10/22)
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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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- Characterization of d-amino acid aminotransferase from Lactobacillus salivarius
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We searched a UniProt database of lactic acid bacteria in an effort to identify d-amino acid metabolizing enzymes other than alanine racemase. We found a d-amino acid aminotransferase (d-AAT) homologous gene (UniProt ID: Q1WRM6) in the genome of Lactobacillus salivarius. The gene was then expressed in Escherichia coli, and its product exhibited transaminase activity between d-alanine and α-ketoglutarate. This is the first characterization of a d-AAT from a lactic acid bacterium. L. salivarius d-AAT is a homodimer that uses pyridoxal-5′-phosphate (PLP) as a cofactor; it contains 0.91 molecules of PLP per subunit. Maximum activity was seen at a temperature of 60 °C and a pH of 6.0. However, the enzyme lost no activity when incubated for 30 min at 30 °C and pH 5.5 to 9.5, and retained half its activity when incubated at pH 4.5 or 11.0 under the same conditions. Double reciprocal plots of the initial velocity and d-alanine concentrations in the presence of several fixed concentrations of α-ketoglutarate gave a series of parallel lines, which is consistent with a Ping-Pong mechanism. The Km values for d-alanine and α-ketoglutarate were 1.05 and 3.78 mM, respectively. With this enzyme, d-allo-isoleucine exhibited greater relative activity than d-alanine as the amino donor, while α-ketobutylate, glyoxylate and indole-3-pyruvate were all more preferable amino acceptors than α-ketoglutarate. The substrate specificity of L. salivarius d-AAT thus differs greatly from those of the other d-AATs so far reported.
- Kobayashi, Jyumpei,Shimizu, Yasuhiro,Mutaguchi, Yuta,Doi, Katsumi,Ohshima, Toshihisa
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- Isolation, purification, and characterization of phenylpyruvate transaminating enzymes of Erwinia carotovora
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Enzymes of Erwinia carotovora that transaminate phenylpyruvate were isolated, purified, and characterized. Two aromatic aminotransferases (PAT1 and PAT2) and an aspartic aminotransferase (PAT3) were found. According to gel filtration, these enzymes have molecular weights of 76, 75, and 78 kDa. The enzymes consist of two identical subunits of molecular weights of 31.4, 31, and 36.5 kDa, respectively. The isoelectric points of PAT1, PAT2, and PAT3 were determined as 3.6, 3.9, and 4.7, respectively. The enzyme preparations considerably differ in substrate specificity. All three of the enzymes productively interacted with the following amino acids: L-aspartic acid, L-leucine (except PAT3), L-isoleucine (except PAT3), L-serine, L-methionine, L-cysteine, L-phenylalanine, L-tyrosine, and L-tryptophane. The aromatic aminotransferases display higher specificity to the aromatic amino acids and the leucine-isoleucine pair, whereas the aspartic aminotransferase displays higher specificity to L-aspartic acid and relatively low specificity to the aromatic amino acids. The aspartic aminotransferase does not use L-leucine or L-isoleucine as a substrate. PAT1, PAT2, and PAT3 show the highest activity at pH 8.9 and at 48, 53, and 58°C, respectively.
- Paloyan,Hambardzumyan,Halebyan
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scheme or table
p. 98 - 104
(2012/06/29)
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- Arg305 of streptomyces l-glutamate oxidase plays a crucial role for substrate recognition
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Recently, we have solved the crystal structure of l-glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 (PDB code: 2E1M), the substrate specificity of which is strict toward l-glutamate. By a docking simulation using l-glutamate and structure of LGOX, we selected three residues, Arg305, His312, and Trp564 as candidates of the residues associating with recognition of l-glutamate. The activity of LGOX toward l-glutamate was significantly reduced by substitution of selected residues with Ala. However, the enzyme, Arg305 of which was substituted with Ala, exhibited catalytic activity toward various l-amino acids. To investigate the role of Arg305 in substrate specificity, we constructed Arg305 variants of LGOX. In all mutants, the substrate specificity of LGOX was markedly changed by the mutation. The results of kinetics and pH dependence on activity indicate that Arg305 of LGOX is associated with the interaction of enzyme and side chain of substrate.
- Utsumi, Tomohiro,Arima, Jiro,Sakaguchi, Chika,Tamura, Takashi,Sasaki, Chiduko,Kusakabe, Hitoshi,Sugio, Shigetoshi,Inagaki, Kenji
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scheme or table
p. 951 - 955
(2012/06/29)
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- Mutant d-amino acid oxidase with higher catalytic efficiency toward d-amino acids with bulky side chains
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d-Amino acid oxidase from the yeast Trigonopsis variabilis (TvDAAO) is widely used in fine organic synthesis, including the preparation of unnatural l-amino acids and α-keto acids. The analysis of the three-dimensional structure of TvDAAO was carried out with the aim of producing the enzyme specific to d-amino acids with bulky side chains. The analysis revealed the residue Phe54 at the entrance to the active site, which controls the substrate access to this site. The residue Phe54 was replaced by residues Ala, Ser, and Tyr. The cultivation of recombinant E. coli strains expressing TvDAAO mutants showed that the mutein with the Phe54Ala substitution had very low stability. Thus, the inactivation of the enzyme occured within 10 min after the cell disruption. The Phe54Ser TvDAAO and Phe54Tyr TvDAAO mutants were obtained as homogeneous preparations, and their thermal stability and catalytic properties were investigated. The introduction of Phe54Ser and Phe54Tyr substitutions resulted in additional stabilization of the protein macromolecule compared to the wild-type TvDAAO. Thus, the half-inactivation time for the mutant enzymes at 54 C increased by a factor of 1.5 and 2, respectively. As in the case of wild-type TvDAAO, the thermal inactivation of the muteins proceeds via a two-step dissociative mechanism. The introduction of mutations led to a strong change in the substrate specificity profile. The mutants have no activity toward a series of d-amino acids (Phe54Ser TvDAAO toward d-Ala, d-Ser, d-Val, and d-Thr; Phe54Tyr TvDAAO toward d-Ser, d-Tyr, d-Thr, and d-Lys). The catalytic efficiency (the k cat/K M ratio) of the Phe54Ser TvDAAO mutant toward d-amino acids with bulky side chains (d-Lys, d-Asn, d-Phe, d-Tyr, d-Trp, and d-Leu) increased from 2.4 to 7.3 times.
- Komarova,Golubev,Khoronenkova,Tishkov
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p. 1489 - 1496
(2013/11/19)
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- Role of the active site residues arginine-216 and arginine-237 in the substrate specificity of mammalian D-aspartate oxidase
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d-Aspartate oxidase (DDO) and d-amino acid oxidase (DAO) are flavin adenine dinucleotide-containing flavoproteins that catalyze the oxidative deamination of d-amino acids. Unlike DAO, which acts on several neutral and basic d-amino acids, DDO is highly specific for acidic d-amino acids. Based on molecular modeling and simulated annealing docking analyses, a recombinant mouse DDO carrying two substitutions (Arg-216 to Leu and Arg-237 to Tyr) was generated (R216L-R237Y variant). This variant and two previously constructed single-point mutants of mouse DDO (R216L and R237Y variants) were characterized to investigate the role of Arg-216 and Arg-237 in the substrate specificity of mouse DDO. The R216L-R237Y and R216L variants acquired a broad specificity for several neutral and basic d-amino acids, and showed a considerable decrease in activity against acidic d-amino acids. The R237Y variant, however, did not show any additional specificity for neutral or basic d-amino acids and its activity against acidic d-amino acids was greatly reduced. The kinetic properties of these variants indicated that the Arg-216 residue is important for the catalytic activity and substrate specificity of mouse DDO. However, Arg-237 is, apparently, only marginally involved in substrate recognition, but is important for catalytic activity. Notably, the substrate specificity of the R216L-R237Y variant differed significantly from that of the R216L variant, suggesting that Arg-237 has subsidiary effects on substrate specificity. Additional experiments using several DDO and DAO inhibitors also suggested the involvement of Arg-216 in the substrate specificity and catalytic activity of mouse DDO and that Arg-237 is possibly involved in substrate recognition by this enzyme. Collectively, these results indicate that Arg-216 and Arg-237 play crucial and subsidiary role(s), respectively, in the substrate specificity of mouse DDO.
- Katane, Masumi,Saitoh, Yasuaki,Maeda, Kazuhiro,Hanai, Toshihiko,Sekine, Masae,Furuchi, Takemitsu,Homma, Hiroshi
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experimental part
p. 467 - 476
(2011/10/05)
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- The role of residues Arg169 and Arg220 in intersubunit interactions of yeast D-Amino acid oxidase
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D-Amino acid oxidase from the yeast Trigonopsis variabilis (EC 1.4.3.3, TvDAAO) exists as a dimer consisting of two identical subunits. The dimeric structure of the enzyme is stabilized by 12 (six pairs) hydrogen bonds, the residues Arg169 and Arg220 of each subunit being involved in eight hydrogen bonds. The Arg169Glu and Arg(169,220)Ala mutants of TvDAAO were prepared. Both mutant enzymes were expressed in E. coli cells as insoluble but catalytically active inclusion bodies. The introduction of amino acid substitutions at the intersubunit interface resulted in a change in the substrate specificity profile and a strong decrease in thermal stability.
- Cherskova,Khoronenkova,Tishkov
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scheme or table
p. 269 - 275
(2011/01/06)
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- PROCESS FOR MAKING N-SULFONATED-AMINO ACID DERIVATIVES
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This invention relates to a process for preparing optically active α -amino acid substrates which are used to make potent lethal factor (LF) inhibitors for the treatment of anthrax. This invention further relates to a process for synthesis of potent LF-inhibitors for the treatment of anthrax. Specifically, the invention concerns a novel, high-yielding and highly enantioselective asymmetric hydrogenation reaction of a tetrasubstituted ene-sulfonamide acid or ester.
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Page/Page column 32
(2008/06/13)
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- Novel substrate specificity of designer 3-isopropylmalate dehydrogenase derived from Thermus thermophilus HB8
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Redesigning of an enzyme for a new catalytic reaction and modified substrate specificity was exploited with 3-isopropylmalate dehydrogenase (IPMDH). Point-mutation on Gly-89, which is not in the catalytic site but near it, was done by changing it to Ala, Ser, Val, and Pro, and all the mutations changed the substrate specificity. The mutant enzymes showed higher catalytic efficiency (kcat/Km) than the native IPMDH when malate was used as a substrate instead of 3-isopropylmalate. More interestingly, an additional insertion of Gly between Gly-89 and Leu-90 significantly altered the substrate-specificity, although the overall catalytic activity was decreased. Particularly, this mutant turned out to efficiently accept D-lactic acid, which was not accepted as a substrate by wild-type IPMDH at all. These results demonstrate the opportunity for creating novel enzymes by modification of amino acid residues that do not directly participate in catalysis, or by insertion of additional residues.
- Fujita, Masaaki,Tamegai, Hideyuki,Eguchi, Tadashi,Kakinuma, Katsumi
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p. 2695 - 2700
(2007/10/03)
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- PROCESS FOR PREPARING SYNTHETIC MATRIX METALLOPROTEASE INHIBITORS
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Synthetic mammalian matrix metalloprotease inhibitors are disclosed that are useful for treating or preventing diseases wherein said diseases are caused by unwanted mammalian matrix metalloprotease activity and include skin disorders, keratoconus, restenosis, rheumatoid arthritis, wounds, cancer, angiogenesis and shock.
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- Concerted base-promoted elimination in the decomposition of N-halo amino acids
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N-Chloroamino acids are unstable in aqueous solution and decompose through different pathways depending on the reaction conditions, yielding precursors of carcinogenic and/or mutagenic compounds. One of these pathways is a 1,2-elimination process, which has scarcely received any attention and for which no systematic analysis is available. The process is first order relative to the N-chloroamino acid and to that of hydroxide ion. The use of 2,2,2-trifluoroethanol and 1,1,1,3,3,3-hexafluoropropan-2-ol buffer solutions established that the process is general-base catalysed. The reaction rate is affected by the presence of a methyl group on the nitrogen atom and the nature of the leaving group. The results show an important steric effect due to the alkyl substituents on the α-carbon. With bulky alkyl substitueras on the α-carbon, and in particular in the case of N-alkylamino acids, the catalytic effect increases as the base strength decreases. To characterize the transition state, Brtonsted's β and βlg were used. A More O'Ferrall-Jencks diagram shows the transition state structure changing from carbanion-like to nitrenium-like, a large perpendicular effect being evident. The reaction proceeds through a concerted mechanism AxhDHDN instead of the stepwise AxhDH? + DN proposed earlier.
- Armesto,Canle L,Garcia,Losada,Santaballa
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p. 552 - 560
(2007/10/03)
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- Palladium-Catalyzed Double and Single Carbonylations of β-Amino Alcohols. Selective Synthesis of Morpholine-2,3-diones and Oxazolidin-2-ones and Applications for Synthesis of α-Oxo Carboxylic Acids
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Catalytic cross double carbonylation of secondary amines and alcohols proceeds in the presence of [PdCl2(MeCN)2] and CuI under carbon monoxide (80 atm) and oxygen (5 atm). Catalytic intramolecular double carbonylation of β-amino alcohols gives morpholine-2,3-diones, which are excellent protecting compounds of amino alcohols and important precursors for biologically active nitrogen compounds. In contrast, catalytic single carbonylation of β-amino alcohols under a mixture (1 : 1) of carbon monoxide and oxygen (1.0 atm) proceeds to give oxazolidin-2-ones selectively. The reaction can be explained by assuming a mechanism which includes intramolecular nucleophilic attack of the hydroxy group of (hydroxyethyl)aminocarbonyl ligands on the CO ligand of the carbamoylpalladium(II) complexes, followed by reductive elimination to give morpholine-2,3-diones. In contrast, direct nucleophilic attack of the hydroxy group to the carbamoyl group affords oxazolidin-2-ones. As a common intermediate for the double and single carbonylations, carbamoylpalladium(II) complex has been isolated by the reaction of [PdCl2(PMe3)2] with β-amino alcohol under CO. The present double carbonylation of amino alcohols provides a novel and convenient method for synthesis of α-oxo carboxylic acids. Thus, the morpholine-2,3-diones obtained undergo reaction with Grignard reagents chemoselectively at the ester positions to give 2-substituted 2-hydroxymorpholin-3-ones, which undergo acid hydrolysis to give α-oxo carboxylic acids.
- Imada, Yasushi,Mitsue, Yo,Ike, Kazuo,Washizuka, Ken-Ichi,Murahashi, Shun-Ichi
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p. 2079 - 2090
(2007/10/03)
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- INHIBITION OF ANGIOGENESIS BY SYNTHETIC MATRIX METALLOPROTEASE INHIBITORS
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Synthetic mammalian matrix metalloprotease inhibitors are useful in controlling angiogenesis. These compounds are thus useful in controlling the growth of tumors and in controlling neovascular glaucomas.
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- MATRIX METALLOPROTEASE INHIBITORS
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Compounds of the formulas STR1 wherein each R 1 is independently H or alkyl (1-8C) and R 2 is alkyl (1-8C) or wherein the proximal R 1 and R 2 taken together are--(CH 2) p--wherein p=3-5;R 3 is H or alkyl (1-4C);R 4 is fused or conjugated unsubstituted or substituted bicycloaryl methylene;n is 0, 1 or 2; m is 0 or 1; andX is OR 5 or NHR 5, wherein R. sup.5 is H or substituted or unsubstituted alkyl (1-12C), aryl (6-12C), aryl alkyl (6-16C); orX is an amino acid residue or amide thereof; orX is the residue of a cyclic amine or heterocyclic amine;wherein R 6 is H or lower alkyl (1-4C) and R 7 is H, lower alkyl (1-4C) or an acyl group, and wherein--CONR 3--is optionally in modified isosteric form are useful for treating conditions which are characterized by unwanted matrix metalloprotease activities.
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- MATRIX METALLOPROTEASE INHIBITORS
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Compounds of the formulas STR1 wherein each R 1 is independently H or alkyl (1-8C) and R 2 is alkyl (1-8C) or wherein the proximal R 1 and R 2 taken together are--(CH 2) p--wherein p=3-5;R 3 is H or alkyl (1-4C);R. sup.4 is fused or conjugated unsubstituted or substituted bicycloaryl methylene;n is 0, 1 or 2; m is 0 or 1; andx is OR 5 or NHR 5, wherein R. sup.5 is H or substituted or unsubstituted alkyl (1-12C), aryl (6-12C), aryl alkyl (6-16C); orX is an amino acid residue or amide thereof; orX is the residue of a cyclic amine or heterocyclic amine;Y is selected from the group consisting of R 7 ONR 6 CONR 6-, R 6 2 NCONOR 7-, and R 6 CONOR. sup.7-, wherein each R 6 is independently H or lower alkyl (1-4C); R 7 is lower alkyl (1-4C) or an acyl group; andwherein--CONR. sup.3--is optionally in modified isoteric form are inhibitors of matrix metalloproteases.
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- Treatment for tissue ulceration
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Compounds of the formulas STR1 wherein R1 is H and R2 is alkyl (3-8C) or wherein R1 and R2 taken together are --(CH2)n -- wherein n=3-5; R3 is H or alkyl (1-4C); R4 is a substituted or unsubstituted fused or conjugated bicycloaryl methylene; X is OR5 or NHR5, wherein R5 is H or substituted or unsubstituted alkyl (1-12C), aryl (6-12C), aryl alkyl (6-16C); or X is an amino acid residue or amide thereof; or X is the residue of a cyclic amine or heterocyclic amine are useful for treating or preventing ulceration of tissue, especially cornea.
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- Isolation and Structure Elucidation of Ergokonin A and B; Two New Antifungal Sterol Antibiotics from Trichoderma koningii
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Isolation and structure elucidation by 2D-NMR spectroscopy of the two new antifungal sterol antibiotics ergokonin A (1) and B (2a) from Trichoderma koningii are described.The structures of 1 and 2a are derived from ergosterol and characterized by an 18-carboxy group.In addition ergokonin A (1) is esterified at the 3-hydroxy group with (2S,3S)-3-hydroxyleucine 3-O-sulfate (3c).In the course of structure elucidation of the amino acid residue the stereoisomers of 3-hydroxyleucine N-sulfamate and 3-O-sulfate (3b, c) have been synthesized and characterized by NMR spectroscopy.
- Augustiniak, Hermann,Forche, Edgar,Reichenbach, Hans,Wray, Viktor,Graefe, Udo,Hoefle, Gerhard
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p. 361 - 366
(2007/10/02)
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- Kinetics of oxidation of amino acids by hexachloroiridate(IV) in aqueous acid medium
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The title reaction studied in the pH range of 2.5 to 3.5 is pseudo-first order in in the presence of excess .The rate increases with increase in and the order in is fractional.The rate also increases with increase in +> and the order in +> is unity.Added salts and change in dielectric constant of the medium do not affect the rate appreciably.However, added acrylamide induces polymerisation.A suitable mechanism has been proposed.
- Kumar, Ch. Sudheer,Chandraiah, U.,Siddiqui, M. A. A.,Kandlikar, Sushma
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p. 714 - 716
(2007/10/02)
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- Pyruvic Acid Dimethylhydrazone. A Synthetic Equivalent of the Pyruvic Acid Dianion
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The pyruvic acid dimethyl hydrazone can be easily prepared in ether.This compound, after deprotonation with alkyllithium, forms a strong nucleophile which on tratment with electrophiles and acidic work up yield α-ketoacids, α-hydroxybutenolides or α,γ-diketoacids.
- Tapia, Ines,Alcazar, Victoria,Moran, Joaquin R.,Caballero, Cruz,Grande, Manuel
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p. 697 - 700
(2007/10/02)
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- 2,3-Dihydro-1,4-dioxin in Organic Chemistry; Part 11. Palladium-Catalyzed Acylations of 5-Tributylstannyl-2,3-dihydro-1,4-dioxin: Preparation of 5-Acyl-2,3-dihydro-1,4-dioxins
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5-Tributylstannyl-2,3-dihydro-1,4-dioxin, readily prepared from 5-lithio-2,3-dihydro-1,4-dioxin and chlorotributyl, smoothly undergoes palladium-catalyzed coupling reactions with acid chlorides, affording 5-acyl-2,3-dihydro-1,4-dioxin in high yield.
- Blanchot, Valerie,Fetizon, Marcel,Hanna, Issam
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p. 755 - 756
(2007/10/02)
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- An Evaluation of the Substrate Specificity, and of Its Modification by Site-Directed Mutagenesis, of the Cloned L-Lactate Dehydrogenase from Bacillus stearothermophilus
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The L-lactate dehydrogenase of Bacillus stearothermophilus (BSLDH) is a stable, thermophilic oxidoreductase.It has been selected as a model of enzymes with considerable future promise in assymetric synthesis in that it has been cloned to ensure a plentiful and inexpensive supply and because of the potential for tailoring its specificity to accept unnatural substrate structures via the site-directed mutagenesis techniques of moleculer biology.In this study, the specificity of BSLDH toward representative α-keto acids possessing straight- and branched-chain alkyl,cycloalkyl, or aromatic side chains has been evaluated.The results show that substrates that are sterically bulky in the region of the α-keto group to be reduced are poorly accepted by the enzyme.Graphics analyses indicated that the low activities of these hindered substrates might be partly due to a bad interaction of the active site residue Gln102 with large or branched substituents adjacent to the α-keto group.Accordingly, Gln102 has been replaced by the smaller Asn residue by site-directed mutagenesis in an attempt to expand the active site volume available to receive substrates larger than the natural pyruvate.However, the kinetic data show that bulky α-keto acids are only marginally better accommodated by the Gln102 -> Asn mutant than by the wild-type enzyme.
- Luyten, Marcel A.,Bur, Daniel,Wynn, Hla,Parris, Wendy,Glod, Marvin,et al.
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p. 6800 - 6804
(2007/10/02)
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- OXIDATION OF 3,4-DISUBSTITUTED ISOXAZOLIN-5-ONES. A NEW SYNTHESIS OF α-KETO ACIDS
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m-Chloroperoxybenzoic or peroxyformic acid oxidation of 3,4-disubstituted isoxazolin-5-ones affords the corresponding 4-hydroxy derivatives from which, by ring opening, nitriles and α-keto acids are obtained.
- Baldoli, Clara,Beccalli, Egle M.,Licandro, Emanuela,Marchesini, Alessandro
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p. 347 - 350
(2007/10/02)
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