- Enantioselective biocatalytic formal α-amination of hexanoic acid to l-norleucine
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A three-step one-pot biocatalytic cascade was designed for the enantioselective formal α-amination of hexanoic acid to l-norleucine. Regioselective hydroxylation by P450CLA peroxygenase to 2-hydroxyhexanoic acid was followed by oxidation to the ketoacid by two stereocomplementary dehydrogenases. Combination with final stereoselective reductive amination by amino acid dehydrogenase furnished l-norleucine in >97% ee.
- Dennig, Alexander,Gandomkar, Somayyeh,Cigan, Emmanuel,Reiter, Tamara C.,Haas, Thomas,Hall, Mélanie,Faber, Kurt
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Read Online
- Structural determinants for the non-canonical substrate specificity of the ω-transaminase from paracoccus denitrificans
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Substrate binding pockets of ω-transaminase (ω-TA) consist of a large (L) pocket capable of dual recognition of hydrophobic and carboxyl substituents, and a small (S) pocket displaying a strict steric constraint that permits entry of a substituent no larger than an ethyl group. Despite the unique catalytic utility of ω-TA enabling asymmetric reductive amination of carbonyl compounds, the severe size exclusion occurring in the S pocket has limited synthetic applications of ω-TA to access structurally diverse chiral amines and amino acids. Here we report the first example of an ω-TA whose S pocket shows a non-canonical steric constraint and readily accommodates up to an n-butyl substituent. The relaxed substrate specificity of the (S)-selective ω-TA, cloned from Paracoccus denitrificans (PDTA), afforded efficient asymmetric syntheses of unnatural amino acids carrying long alkyl side chains such as lnorvaline and l-norleucine. Molecular modeling using the recently released X-ray structure of PDTA could pinpoint an exact location of the S pocket which had remained dubious. Entry of a hydrophobic substituent in the L pocket was found to have the S pocket accept up to an ethyl substituent, reminiscent of the canonical steric constraint. In contrast, binding of a carboxyl group to the L pocket induced a slight movement of V153 away from the small-pocketforming residues. The resulting structural change elicited excavation of the S pocket, leading to formation of a narrow tunnel-like structure allowing accommodation of linear alkyl groups of carboxylatebearing substrates. To verify the active site model, we introduced site-directed mutagenesis to six active site residues and examined whether the point mutations alleviated the steric constraint in the S pocket. Consistent with the molecular modeling results, the V153A variant assumed an elongated S pocket and accepted even an n-hexyl substituent. Our findings provide precise structural information on substrate binding to the active site of ω-TA, which is expected to benefit rational redesign of substrate specificity of ω-TA.
- Park, Eul-Soo,Park, Sae-Rom,Han, Sang-Woo,Dong, Joo-Young,Shin, Jong-Shik
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Read Online
- 2(S)-Aminohex-5-ynoic acid, an antimetabolite from Cortinarius claricolor var. Tenuipes
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Screening for antimetabolites in edible mushrooms showed that the hot water extract of fruiting bodies of Cortinarius claricolor var. tenuipes strongly inhibited the growth of Bacillus subtilis B-50 in a chemically defined minimal medium. 2(S)-Aminohex-5-ynoic acid was isolated as an active compound.
- Aoyagi, Yasuo,Sugahara, Tatsuyuki
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Read Online
- Semi-rational hinge engineering: modulating the conformational transformation of glutamate dehydrogenase for enhanced reductive amination activity towards non-natural substrates
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The active site is the common hotspot for rational and semi-rational enzyme activity engineering. However, the active site represents only a small portion of the whole enzyme. Identifying more hotspots other than the active site for enzyme activity engineering should aid in the development of biocatalysts with better catalytic performance. Glutamate dehydrogenases (GluDHs) are promising and environmentally benign biocatalysts for the synthesis of valuable chirall-amino acids by asymmetric reductive amination of α-keto acids. GluDHs contain an inter-domain hinge structure that facilitates dynamic reorientations of the domains relative to each other. Such hinge-bending conformational motions of GluDHs play an important role in regulating the catalytic activity. Thus, the hinge region represents a potential hotspot for catalytic activity engineering for GluDHs. Herein, we report semi-rational activity engineering of GluDHs with the hinge region as the hotspot. Mutants exhibiting significantly improved catalytic activity toward several non-natural substrates were identified and the highest activity increase reached 104-fold. Molecular dynamics simulations revealed that enhanced catalytic activity may arise from improving the open/closed conformational transformation efficiency of the protein with hinge engineering. In the batch production of three valuablel-amino acids, the mutants exhibited significantly improved catalytic efficiency, highlighting their industrial potential. Moreover, the catalytic activity of several active site tailored GluDHs was also increased by hinge engineering, indicating that hinge and active site engineering are compatible. The results show that the hinge region is a promising hotspot for activity engineering of GluDHs and provides a potent alternative for developing high-performance biocatalysts toward chirall-amino acid production.
- Liu, Yayun,Meng, Lijun,Wu, Jianping,Yang, Lirong,Yin, Xinjian,Zhou, Haisheng
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p. 3376 - 3386
(2020/06/09)
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- 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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- Preparative Asymmetric Synthesis of Canonical and Non-canonical a-amino Acids through Formal Enantioselective Biocatalytic Amination of Carboxylic Acids
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Chemical and biocatalytic synthesis of non-canonical a-amino acids (ncAAs) from renewable feedstocks and using mild reaction conditions has not efficiently been solved. Here, we show the development of a three-step, scalable and modular one-pot biocascade for linear conversion of renewable fatty acids (FAs) into enantiopure l-a-amino acids. In module 1, selective a-hydroxylation of FAs is catalyzed by the P450 peroxygenase P450CLA. By using an automated H2O2 supplementation system, efficient conversion (46 to >99%; TTN>3300) of a broad range of FAs (C6:0 to C16:0) into valuable a-hydroxy acids (a-HAs; >90% a-selective) is shown on preparative scale (up to 2.3 gL1 isolated product). In module 2, a redox-neutral hydrogen borrowing cascade (alcohol dehydrogenase/amino acid dehydrogenase) allowed further conversion of a-HAs into l-a-AAs (20 to 99%). Enantiopure l-a-AAs (e.e. >99%) including the pharma synthon l-homo-phenylalanine can be obtained at product titers of up to 2.5 gL1. Based on renewables and excellent atom economy, this biocascade is among the shortest and greenest synthetic routes to structurally diverse and industrially relevant ncAAs.
- Dennig, Alexander,Blaschke, Fabio,Gandomkar, Somayyeh,Tassano, Erika,Nidetzky, Bernd
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p. 1348 - 1358
(2019/10/28)
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- Combinatorial Mutation Analysis of ω-Transaminase to Create an Engineered Variant Capable of Asymmetric Amination of Isobutyrophenone
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ω-Transaminase (ω-TA) is an important enzyme for asymmetric synthesis of chiral amines. Rapid creation of a desirable ω-TA variant, readily available for scalable process operation, is demanded and has attracted intense research efforts. In this study, we aimed to develop a quantitative mutational analysis (i. e., R-analysis) that enables prediction of combinatorial mutation outcomes and thereby provides reliable guidance of enzyme engineering through combination of already characterized mutations. To this end, we determined three mutatable active-site residues of ω-TA from Ochrobactrum anthropi (i. e., leucine 57, tryptophan 58 and valine 154) by examining activities of nine alanine-scanning mutants for seven substrate pairs. The R-analysis of the mutatable residues is based on assessment of changes in relative activities for a series of structurally analogous substrates. Using three sets of substrates (five α-keto acids, six arylalkylamines and three arylalkyl ketones), we found that combination of two point mutations display additive effects of each mutational outcome such as steric relaxation for bulky substrates or catalytic enhancement for amination of ketones. Consistent with the R-analysis-based prediction, the ω-TA variant harboring triple alanine mutations, i. e. L57A, W58A and V154A, showed high activity improvements for bulky substrates, e. g. a 3.2×104-fold activity increase for 1-phenylbutylamine. The triple mutant even enabled asymmetric amination of isobutyrophenone, carrying a branched-chain alkyl substituent to be accepted in a small binding pocket that normally shows a steric limit up to an ethyl group, with >99% ee of a resulting (S)-amine. (Figure presented.).
- Kim, Hong-Gon,Han, Sang-Woo,Shin, Jong-Shik
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p. 2594 - 2606
(2019/05/15)
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- Preparative Asymmetric Synthesis of Canonical and Non-canonical α-amino Acids Through Formal Enantioselective Biocatalytic Amination of Carboxylic Acids
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Chemical and biocatalytic synthesis of non-canonical α-amino acids (ncAAs) from renewable feedstocks and using mild reaction conditions has not efficiently been solved. Here, we show the development of a three-step, scalable and modular one-pot biocascade for linear conversion of renewable fatty acids (FAs) into enantiopure l-α-amino acids. In module 1, selective α-hydroxylation of FAs is catalyzed by the P450 peroxygenase P450CLA. By using an automated H2O2 supplementation system, efficient conversion (46 to >99%; TTN>3300) of a broad range of FAs (C6:0 to C16:0) into valuable α-hydroxy acids (α-HAs; >90% α-selective) is shown on preparative scale (up to 2.3 g L?1 isolated product). In module 2, a redox-neutral hydrogen borrowing cascade (alcohol dehydrogenase/amino acid dehydrogenase) allowed further conversion of α-HAs into l-α-AAs (20 to 99%). Enantiopure l-α-AAs (e.e. >99%) including the pharma synthon l-homo-phenylalanine can be obtained at product titers of up to 2.5 g L?1. Based on renewables and excellent atom economy, this biocascade is among the shortest and greenest synthetic routes to structurally diverse and industrially relevant ncAAs. (Figure presented.).
- Dennig, Alexander,Blaschke, Fabio,Gandomkar, Somayyeh,Tassano, Erika,Nidetzky, Bernd
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supporting information
(2019/02/09)
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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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- Nitrilases
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The invention relates to nitrilases and to nucleic acids encoding the nitrilases. In addition, methods of designing new nitrilases and methods of use thereof are also provided. The nitrilases have increased activity and stability at increased pH and temperature.
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Paragraph 0495
(2015/09/22)
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- Deracemization of amino acids by coupling transaminases of opposite stereoselectivity
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Biocatalytic deracemization of amino acids without relying on oxidase-based deamination of an unwanted enantiomer was demonstrated by coupling a-and w-transaminases displaying opposite stereoselectivity. This strategy employs isopropylamine and a keto acid as cosubstrates and is free of generation of hydrogen peroxide which is troublesome in the conventional oxidase-based methods.
- Park, Eul-Soo,Shin, Jong-Shik
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p. 3505 - 3509
(2015/02/19)
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- 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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- Amidohydrolase Process: Expanding the use of l-N-carbamoylase/N-succinyl- amino acid racemase tandem for the production of different optically pure l-amino acids
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A bienzymatic system comprising an N-succinylamino acid racemase from Geobacillus kaustophilus CECT4264 (GkNSAAR) and an enantiospecific l-N-carbamoylase from Geobacillus stearothermophilus CECT43 (BsLcar) has been developed. This biocatalyst has been able to produce optically pure natural and non-natural l-amino acids starting from racemic mixtures of N-acetyl-, N-formyl- and N-carbamoyl-amino acids by dynamic kinetic resolution. The fastest conversion rate was found with N-formyl-amino acids, followed by N-carbamoyl- and N-acetyl-amino acids, and GkNSAAR proved to be the limiting step of the system due to its lower specific activity. Metal ion cobalt was essential for the activity of the biocatalyst and the system was optimally active when Co 2+ was added directly to the reaction mixture. The optimum pH for the biocatalyst proved to be 8.0, for both N-formyl- and N-carbamoyl-amino acid substrates, whereas optimum temperature ranges were 45-55 °C for N-formyl-amino acids and 55-70 °C for N-carbamoyl-derivatives. The bienzymatic system was equally efficient in converting aromatic and aliphatic substrates. Total conversion was also achieved using high substrate concentrations (100 and 500 mM) with no noticeable inhibition. This "Amidohydrolase Process" enables the production of both natural and non-natural l-amino acids from a broad substrate spectrum with yields of over 95%.
- Soriano-Maldonado, Pablo,Rodríguez-Alonso, María José,Hernández-Cervantes, Carmen,Rodríguez-García, Ignacio,Clemente-Jiménez, Josefa María,Rodríguez-Vico, Felipe,Martínez-Rodríguez, Sergio,Las Heras-Vázquez, Francisco Javier
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p. 1281 - 1287
(2014/07/22)
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- 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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- SEPARATING AGENT FOR CHROMATOGRAPHY
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A separating agent for chromatography is provided that is useful for the separation of specific compounds, e.g., for the optical resolution of amino acids. This separating agent for chromatography provides a higher productivity and contains a crown ether-like cyclic structure and optically active binaphthyl. This separating agent for chromatography containing a crown ether-like cyclic structure and optically active binaphthyl is provided by introducing a substitution group for binding to carrier into a specific commercially available 1,1′-binaphthyl derivative that has substituents at the 2, 2′, 3, and 3′ positions, then introducing a crown ether-like cyclic structure, and subsequently chemically bonding the binaphthyl derivative to the carrier through the substitution group for binding to carrier.
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Paragraph 0074; 0075
(2013/08/15)
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- Thailandepsins: Bacterial products with potent histone deacetylase inhibitory activities and broad-spectrum antiproliferative activities
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Histone deacetylase (HDAC) inhibitors have emerged as a new class of anticancer drugs, with one synthetic compound, SAHA (vorinostat, Zolinza; 1), and one natural product, FK228 (depsipeptide, romidepsin, Istodax; 2), approved by FDA for clinical use. Our studies of FK228 biosynthesis in Chromobacterium violaceum no. 968 led to the identification of a cryptic biosynthetic gene cluster in the genome of Burkholderia thailandensis E264. Genome mining and genetic manipulation of this gene cluster further led to the discovery of two new products, thailandepsin A (6) and thailandepsin B (7). HDAC inhibition assays showed that thailandepsins have selective inhibition profiles different from that of FK228, with comparable inhibitory activities to those of FK228 toward human HDAC1, HDAC2, HDAC3, HDAC6, HDAC7, and HDAC9 but weaker inhibitory activities than FK228 toward HDAC4 and HDAC8, the latter of which could be beneficial. NCI-60 anticancer screening assays showed that thailandepsins possess broad-spectrum antiproliferative activities with GI50 for over 90% of the tested cell lines at low nanomolar concentrations and potent cytotoxic activities toward certain types of cell lines, particularly for those derived from colon, melanoma, ovarian, and renal cancers. Thailandepsins thus represent new naturally produced HDAC inhibitors that are promising for anticancer drug development.
- Wang, Cheng,Henkes, Leonhard M.,Doughty, Leah B.,He, Min,Wang, Difei,Meyer-Almes, Franz-Josef,Cheng, Yi-Qiang
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supporting information; experimental part
p. 2031 - 2038
(2011/12/15)
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- Asymmetric synthesis of allylic secondary alcohols: Anewgeneral approach for the preparation of α-amino acids
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A new general approach for the synthesis of optically active a-amino acids has been developed.The key steps involve a ruthenium catalysed cross-coupling reaction to give a range of α,β-unsaturated ketones, which were then reduced to allylic secondary alcohols in the presence of a chiral CBS oxazaborolidine.A thermal Overman rearrangement was used to prepare a series of allylic trichloroacetimidates and these were converted under standard conditions to the target α-amino acids in good overall yields.
- Drummond, Lorna J.,Sutherland, Andrew
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experimental part
p. 5349 - 5356
(2010/08/06)
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- Simplifying pyridoxal: Practical methods for amino acid dynamic kinetic resolution
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Figure presented Metal complexes of picolinaldehyde are identified as low-cost and environmentally benign catalysts, providing high reaction rates and turnovers for the racemization of amino acids. These pyridoxal surrogates demonstrate activity toward a variety of amino acid esters. Applications to chemoenzymatic dynamic kinetic resolutions provide access to amino acids in high yields and with excellent enantioselectivities, demonstrating their compatibility with protease-mediated transformations.
- Felten, Albert E.,Zhu, Gangguo,Aron, Zachary D.
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supporting information; experimental part
p. 1916 - 1919
(2010/07/06)
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- Resolution of non-protein amino acids via the microbial protease-catalyzed enantioselective hydrolysis of their N-unprotected esters
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In the Aspergillus oryzae protease-catalyzed ester hydrolysis, substitution of N-unprotected amino acid esters for the corresponding N-protected amino acid esters resulted in a large enhancement of the hydrolysis rate, while the enantioselectivity was deteriorated strikingly when the substrates employed were the conventional methyl esters. This difficulty was overcome by employing esters bearing a longer alkyl chain such as the isobutyl ester. Utilizing this ester, amino acids carrying an aromatic side chain were resolved with excellent enantioselectivities (E=50 to >200). With amino acids bearing an aliphatic side chain also, good results in terms of the hydrolysis rate and enantioselectivity were obtained by employing such an ester as the isobutyl ester. Moreover, the enantioselectivity proved to be enhanced further by conducting the reaction at low temperature. This procedure was applicable to the case where the enantioselectivity was not high enough even by the use of the isobutyl ester.
- Miyazawa, Toshifumi,Imagawa, Kiwamu,Minowa, Hiroe,Miyamoto, Toyoko,Yamada, Takashi
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p. 10254 - 10261
(2007/10/03)
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- Highly diastereoselective alkylation of the Dellaria oxazinone template with bifunctional electrophiles
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This study investigates the efficiency of alkylation of the Dellaria oxazinone glycinate template with sensitive bifunctional electrophiles. In addition to improved access to the template, triflate/halide bifunctional combinations provided good yields (85-93%) of highly diastereoselective alkylation products.
- Roos, Gregory H. P.,Dastlik, Kim A.
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p. 2197 - 2208
(2007/10/03)
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- Dynamic kinetic resolution of racemic N-phthalyl amino acids using (S)-α-methylpantolactone as the chiral auxiliary
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A dynamic kinetic resolution of racemic N-phthalyl amino acids by stereoselective esterification was examined using (S)-α-methylpantolactone as the chiral auxiliary. The reaction of various racemic N-phthalyl amino acids with this chiral alcohol in the presence of both DCC and DMAP afforded predominantly the (S,S)-esters in nearly quantitative yield. Copyright (C) 2000 Elsevier Science Ltd.
- Calmes, Monique,Glot, Christele,Michel, Thibaut,Rolland, Marc,Martinez, Jean
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p. 737 - 741
(2007/10/03)
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- Chemo-enzymatic synthesis of optically active amino acids and peptides
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The industrial alkaline protease, alcalase, is stable and active in a high concentration of organic solvents and useful as a biocatalyst for (i) diastereoselective hydrolysis of peptide esters and preparation of racemization-free peptides; (ii) selective incorporation of esters of D-amino acid into peptides in t-butanol via a selective hydrolysis of esters of D,L-amino acid, followed by using the unhydrolyzed D-esters as a nucleophile in a kinetically controlled peptide bond formation; (iii) resolution of esters of amino acid in 95% t-butanol/5% water, followed by saponification of the unreacted esters to offer both enantiomers with high yield and optical purity; (iv) completely resolve amino-acid esters with high yield and optical purity via in situ racemization of the unreacted antipode catalyzed by pyridoxal 5-phosphate; (v) cryobioorganic synthesis of peptides with increased yields 15%-40% of peptide bond formation by reaction at 5 °C instead of 25-30 °C of a kinetically controlled enzymatic reaction in alcohols.
- Chen, Shui-Tein,Wang, Kung-Tsung
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p. 301 - 311
(2007/10/03)
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- Acylase I-catalyzed deacetylation of N-acetyl-L-cysteine and S-alkyl-N- acetyl-L-cysteines
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The aminoacylase that catalyzes the hydrolysis of N-acetyl-L-cysteine (NAC) was identified as acylase I after purification by column chromatography and electrophoretic analysis. Rat kidney cytosol was fractionated by ammonium sulfate precipitation, and the proteins were separated by ion-exchange column chromatography, gel-filtration column chromatography, and hydrophobic interaction column chromatography. Acylase activity with NAC and N-acetyl-L- methionine (NAM), a known substrate for acylase I, as substrates coeluted during all chromatographic steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the protein was purified to near homogeneity and had a subunit M(r) of 43 000, which is identical with the M(r) of acylase I from porcine kidney and bovine liver. n-Butylmalonic acid was a slow-binding inhibitor of acylase I and inhibited the deacetylation of NAC with a K(i) of 192 ± 27 μM. These results show that acylase I catalyzes the deacetylation of NAC. The acylase I-catalyzed deacetylation of a range of S-alkyl-N- acetyl-L-cysteines, their carbon and oxygen analogues, and the selenium analogue of NAM was also studied with porcine kidney acylase I. The specific activity of the acylase I-catalyzed deacetylation of these substrates was related to their calculated molar volumes and log P values. The S-alkyl-N- acetyl-L-cysteines with short (C0-C3) and unbranched S-alkyl substituents were good acylase I substrates, whereas the S-alkyl-N-acetyl-L-cysteines with long (>C3) and branched S-alkyl substituents were poor acylase I substrates. The carbon and oxygen analogues of S-methyl-N-acetyl-L-cysteine and the carbon analogue of S-ethyl-N-acetyl-L-cysteine were poor acylase I substrates, whereas the selenium analogue of NAM was a good acylase I substrate.
- Uttamsing, Vinita,Keller,Anders
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p. 800 - 809
(2007/10/03)
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- Substrate recognition and saturation kinetics in de novo designed histidine-based four-helix bundle catalysts
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Designed four-helix bundle proteins with reactive sites based on the cooperativity of HisH+-His pairs in helical sequences catalyze acyl-transfer reactions of p-nitrophenyl esters with large rate enhancements. The function of the HisH+/su
- Broo, Kerstin S.,Nilsson, Helena,Nilsson, Jonas,Baltzer, Lars
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p. 10287 - 10295
(2007/10/03)
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- Resolution of non-protein amino acids via microbial protease-catalyzed ester hydrolysis: Marked enhancement of enantioselectivity by the use of esters with longer alkyl chains and at low temperature
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In the microbial protease-catalyzed hydrolysis of amino acid esters with the free α-amino group, the enantioselectivity can be enhanced greatly by employing esters with longer alkyl chains such as the isobutyl ester instead of the conventional methyl ester and by conducting the reaction at low temperature.
- Miyazawa, Toshifumi,Minowa, Hiroe,Miyamoto, Toyoko,Imagawa, Kiwamu,Yanagihara, Ryoji,Yamada, Takashi
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p. 367 - 370
(2007/10/03)
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- L-Methionine related 1-amino acids by acylase cleavage of their corresponding N-acetyl-DL-derivatives
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Acylase I from Aspergillus oryzae is an even more useful enzyme than suggested so far. Besides standard amino acids such as L-Met, L-Val and L-Phe, a number of additional sulfur- and selenium-containing amino acids can be obtained at useful reaction rates and in very high enantiomeric purity by kinetic resolution of the respective N-acetyl-DL-amino acids.
- Bommarius, Andreas S.,Drauz, Karlheinz,Guenther, Kurt,Knaup, Guenter,Schwarm, Michael
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p. 3197 - 3200
(2007/10/03)
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- 2-oxoethyl derivatives as immunosuppressants
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A class of compounds that suppress human T-lymphocyte proliferation is disclosed. The active compounds essentially contain at least the following structure: STR1
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- Asymmetric synthesis of α-amino acids via diastereoselective addition of (R)-pantolactone to their ketenes
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The diastereoselective addition of (R)-pantolactone to various amino ketenes derived from phthalylamino acids is reported. The configuration of the newly-generated asymmetric center is dependent on alkyl or aryl C(x substitution. This method constitutes a novel and convenient way of amino acid deracemization.
- Calmes, Monique,Daunis, Jacques,Mai, Nathalie
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p. 1641 - 1648
(2007/10/03)
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- Asymmetric alkylations of a sultam-derived glycine equivalent: Practical preparation of enantiomerically pure α-amino acids
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Alkylation of the chiral glycine derivative 2 with 'activated' organohalides under ultrasound-assisted phase-transfer catalysis or with activated and nonactivatcd organohalides in anhydrous medium provides (mostly crystalline) alkylation products 3. Acidic hydrolysis of the pure products 3 gives (aminoacyl)sultams 4 which by mild saponification furnish pure α-amino acids 5 in good overall yields from 2, along with recovered auxiliary 1 (Scheme 1). Pure ω-protected α,ω-diamino acids and α-amino-ω-(hydroxyamino)acids 12-16 are readily accessible from (ω-haloacyl)sultams 3 via reaction with N-nucleophiles followed by acidic and basic hydrolyses (Scheme 2). A reliable determination of the enantiomeric purity of α-amino acids using HPLC analysis of their N-(3,5-dinitrobenzoyl)prolyl derivatives 17 is presented.
- Oppolzer,Moretti,Zhou
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p. 2363 - 2380
(2007/10/02)
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- Optical resolution, characterization, and X-ray crystal structures of diastereomeric salts of chiral amino acids with (S)-(-)-1-phenylethanesulfonic acid
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Ten DL-Amino acids (AA), including neutral, and basic amino acids, and an imino acid, were optically resolved, without derivatization into their covalent compounds, by means of fractional crystallization of their diastereomeric salts with (-)-1-phenylethanesulfonic acid (PES) in various solvents. Several pairs of the diastereomeric crystalline salts formed during the resolutions were analyzed by DSC and spectroscopy, which showed that the successful resolutions were attributable to differences in various physicochemical properties between the more-soluble D-AA · (-)-PES and less-soluble L-AA · (-)-PES. Chiral recognition of the most successfully resolved species, DL-p-hydroxyphenylglycine (HPG) salt, was explored by comparing the X-ray crystal structures of D- and L-HPG · (-)-PES. The two crystal structures differed obviously in their hydrogen-bonding networks: the less-soluble L-HPG · (-)-PES only had strong hydrogen-bonded infinite chains of HPG in a 'head-to-tail' arrangement through the p-hydroxyl group, the structure of which was more geometrically stable than that of the more-soluble D-HPG · (-)-PES. The differences in the two crystal structures related to striking differences in their solubilities and thermal properties.
- Yoshioka,Ohtsuki,Da-Te,Okamura,Senuma
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p. 3012 - 3020
(2007/10/02)
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- Resolution of amino acids in a mixture of 2-methyl-2-propanol/water (19:1) catalyzed by alcalase via in situ racemization of one antipode mediated by pyridoxal 5-phosphate
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Procedures for the conversion of a racemic amino acid into the L-enantiomer by the alcalase catalyzed resolution of the amino acid ester in 2-methyl-2-propanol/water (19:1) simultaneously with the pyridoxal 5-phosphate-catalyzed racemization of the unhydrolyzed antipode have been developed.
- Chen,Huang,Wang
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p. 7580 - 7581
(2007/10/02)
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- Amino Acid Synthesis via Ring Opening of N-Sulphonyl Aziridine-2-Carboxylate Esters with Organometallic Reagents.
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Nucleophilic ring opening of optically active N-sulphonyl aziridine-2-carboxylate esters with organometallic reagents has been investigated as a method of preparation of optically active amino acids.Key Words: aziridine-2-carboxylate, cuprate, nucleophilic ring opening, amino acid
- Baldwin, Jack E.,Spivey, Alan C.,Schofield, Christopher J.,Sweeney, Joseph B.
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p. 6309 - 6330
(2007/10/02)
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- Calorimetric Evaluation of Enzyme Kinetic Parameters
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The measurement of kinetic parameters (kcat, Km, Ki) for a wide range of proteolytic enzymes is vital to contemporary bioorganic and medicinal chemistry.Enzyme assays based on changes in optical properties of the system or changes in concentration of an ion detectable electrochemically are not viable for many enzyme-catalyzed reactions, including proteases and peptidases.Hydrolysis of an amide bond produces no change in the optical properties or pH of the reaction solution, and as a result no general direct method for the evaluation of protease kinetics exists using underivatized substrates.We report here a microcalorimetric assay which provides a general and straightforward technique for the measurement of kinetic parameters of hydrolysis of underivatized peptide substrates by proteases.Using this technique, kcat values as high as 105 s-1 can be easily measured.We demonstrate the utility of the technique by measuring the kinetics of hydrolysis of several N-acylamino acids by the synthetically useful enzyme hog kidney acylase and the hydrolysis of tetrapeptide p-nitrophenyl anilides by subtilisin BPN'.Although we have used the technique to monitor amide bond hydrolysis, the methodology is applicable to any system with appropriate kinetic and thermodynamic properties.
- Williams, Brent A.,Toone, Eric J.
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p. 3507 - 3510
(2007/10/02)
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- Asymmetric synthesis X III: The stereocontrolled synthesis of (R)-α-amino acids via a double chiral induction
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The stereocontrolled synthesis of (R)-α-amino acids via a double chiral induction in alkylation of the ketimine derived from (+)-camphor and (+)-methyl glycinate which is a chiral match pair has been studied. (R)-α-amino acids with high optical purity 80-90% are obtained after hydrolysis of the alkylated products. We find that the chiral match which is the synergistic effect in a double chiral induction is very important to obtain the higher diastereoselectivity for the stereocontrolled synthesis.
- Yao Zhong,Guilan,Changyou,Huri,Lanjun,Aiqiao
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p. 1087 - 1090
(2007/10/02)
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- Non-Proteinogenic Amino Acid Synthesis: Synthesis of β,γ-Unsaturated α-Amino Acids from Aspartic Acid
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A general, stereospecific, synthesis of β,γ-unsaturated α-amino acids using the β-anion derived from aspartic acid is described.Keywords: aspartic acid; unsaturated acids
- Baldwin, Jack E.,Moloney, Mark G.,North, Michael
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p. 6319 - 6330
(2007/10/02)
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- ASYMMETRIC ALKYLATIONS OF A SULTAM-DERIVED GLYCINATE EQUIVALENT: PRACTICAL PREPARATION OF ENANTIOMERICALLY PURE α-AMINO ACIDS
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Deprotonation/alkylation of sultam-derived N-glycinate equivalent 3 gave crystalline products 5 which on mild hydrolysis furnished α-amino acids 7 (ca.100percent e.e.) in high overall yield.
- Oppolzer, Wolfgang,Moretti, Robert,Thomi, Silvia
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p. 6009 - 6010
(2007/10/02)
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- The Ring Opening of Aziridine-2-carboxylate Esters with Organometallic Reagents
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The ring opening of aziridines with organocuprate reagents provides a new entry to amino acids.
- Baldwin, Jack E.,Adlington, Robert M.,O'Neil, Ian A.,Schofield, Christopher,Spivey, Alan C.,Sweeney, Joseph B.
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p. 1852 - 1854
(2007/10/02)
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- NEW CHIRAL SYNTHON OF ELECTROPHILIC GLYCINE AND ITS REACTIONS WITH METALLOORGANIC, C-H, N-H, AND O-H COMPOUNDS FOR THE ASYMMETRIC SYNTHESIS OF α-SUBSTITUTED α-AMINOACIDS
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A new chiral synthon of the electrophilic glycine - the Ni(II) complex of the Schiff base of L-N-benzylprolylorthoaminobenzophenone with α-bromoglycine - was synthesized.The reaction of this complex with nucleophiles led to the isolation of complexes of L- and D-2-amino-2-dimethylaminoacetic acid, L- and D-2-amino-2-phenoxyacetic acid, 3,3-di(carbethoxy)alanine, and norleucine.The decomposition of the 3,3-di(carbethoxy)alanine and norleucine complexes led to the synthesis of L-aspartic acid and L-norleucine with the enantiomeric purity of 80 and 68 percent correspondingly.
- Belokon', Yu. N.,Popkov, A. N.,Chernoglazova, N. I.,Bakhmutov, V. I.,Saporovskaya, M. B.,Belikov, V. M.
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p. 1744 - 1748
(2007/10/02)
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- GENERAL METHOD FOR THE ASYMMETRIC SYNTHESIS OF α-AMINO ACIDS VIA ALKYLATION OF THE CHIRAL NICKEL(II) SCHIFF BASE COMPLEXES OF GLYCINE AND ALANINE
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Nickel(II) complexes of Schiff bases derived from (S)-o-benzaldehyde and alanine (3), or (S)-O-benzophenone and alanine (4), or glycine (5) have been used for the asymmetric synthesis of α-amino acids under a variety of conditions.The method of choice consists of the reaction of the corresponding complex with the appropriate alkyl halide in DMF at 25 deg C using solid NaOH as a catalyst.Low diastereoselective excess (d.e.) is observed for the alkylation of complex (3) with benzyl bromide and allyl bromide.Large selectivity (80 percent) is observed for the alkylation of complex (4).Optically pure(R)- and (S)-O-benzyl-α-methyl-α-amino acids were obtained (70-90 percent) after the alkylated diastereoisomeric complexes had been seperated on SiO2 and hydrolysed with aqueous HCl.The initial chiral reagents were recovered (80-92 percent).The alkylation of complex (5) gave (S)-alanine, (S)-valine, (S)-phenylalanine, (S)-tryptophan, (S)-isoleucine, (S)-2-aminohexanoic acid, and 3,4-dimethoxyphenylalanine with optical yields of 70-92 percent.The optically pure α-amino acids were obtained after the separation of the alkylated diastereoisomeric complexes on SiO2.The stereochemical mechanism of the alkylation reaction is discussed.
- Belokon, Yuri N.,Bakhmutov, Vladimir I.,Chernoglazova, Nina I.,Kochetkov, Konstantin A.,Vitt, Sergei V.,et al.
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p. 305 - 312
(2007/10/02)
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- Synthesis of a Chiral Nickel(II) Complex of an Electrophilic Glycinate, and its Use for Asymmetric Preparation of α-Amino Acids
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A chiral NiII complex of a Schiff's base derived from (S)-o-benzophenone and α-bromoglycine has been obtained and its stereoselective reaction with nucleophiles studied; the synthesis of aspartic acid with 80percent optical purity is described.
- Belokon', Yuri N.,Popkov, Aleksander N.,Chernoglazova, Nina I.,Saporovskaya, Marina B.,Bakhmutov, Vladimir I.,Belikov, Vasili M.
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p. 1336 - 1338
(2007/10/02)
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- NEW SYNTHESES OF α-AMINO ACIDS BASED ON N-ACYLIMINO ACETATES
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The reaction of N-acylamino-2-bromoacetates 2 ( via N-acylimino acetates 3 ) with higher order mixed cuprates, trimethylsilyl enol ethers and β-dicarbonyl compounds leads to a variety of α-amino acid derivatives.Their conversion into the free amino acids can be conveniently carried out by the use of t-butyl protection.In case of the N-acetyl compounds cleavage of the protecting group and optical resolution can be achieved in one step hog renal acylase.
- Bretschneider, Thomas,Miltz, Wolfgang,Muenster, Peter,Steglich, Wolfgang
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p. 5403 - 5414
(2007/10/02)
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- Practical Asymmetric Syntheses of α-Amino Acids through Carbon-Carbon Bond Constructions on Electrophilic Glycine Templates
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The optically active D- and L-erythro-4-(benzyloxycarbonyl)-5,6-diphenyl-2,3,5,6-tetrahydro-4H-1,4-oxazin-2-ones (3) and D- and L-erythro-4-(tert-butoxycarbonyl)-5,6-diphenyl-2,3,5,6-tetrahydro-4H-1,4-oxazin-2-ones (3) can be efficiently brominated to serve as electrophilic glycine templates for the asymmetric synthesis of amino acids.It was found that coupling to these templates can proceed with either net retention or net inversion of stereochemistry.The final deblocking to the amino acids is accomplished with either dissolving-metal reduction or catalytic hydrogenolysis.The syntheses of β-ethyl aspartic acid, norvaline, allylglycine, alanine, norleucine, homophenylalanine, p-methoxyhomophenylalanine, cyclopentylglycine, and cyclopentenylglycine and a formal synthesis of clavalanine are described.In addition, the direct asymmetric syntheses of N-t-BOC-allylglycine and N-t-BOC-cyclopentenylglycine are described.
- Williams, Robert M.,Sinclair, Peter J.,Zhai, Dongguan,Chen, Daimo
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p. 1547 - 1557
(2007/10/02)
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- CYCLOPROPYLALANINE, AN ANTIFUNGAL AMINO ACID OF THE MUSHROOM AMANITA VIRGINEOIDES BAS
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Cyclopropylalanine, an antifungal amino acid, has been isolated from the mushroom Amanita virgineoides Bas.Its structure has been elucidated as (2s)-2-amino-3-cyclopropylpropionic acid, on the basis of the spectroscopic analysis and its synthesis from L-allylglycine.
- Ohta, Tomihisa,Nakajima, Shigeru,Sato, Zenji,Aoki, Toshio,Hatanaka, Shin-ichi,Nozoe, Shigeo
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p. 511 - 512
(2007/10/02)
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- ASYMMETRIC SYNTHESIS OF α-AMINO ACIDS FROM α-HALOGENATED 10-SULFONAMIDO-ISOBORNYL ESTERS.
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Treatment of chiral α-haloesters 2 with NaN3 gave azidoesters 3 wich on successive transesterification and hydrogenolysis furnished α-amino acids 5 and 9 in high e.e.
- Oppolzer, Wolfgang,Pedrosa, Rafael,Moretti, Robert
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p. 831 - 834
(2007/10/02)
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