632-20-2Relevant articles and documents
Single-Cell-Based Screening and Engineering of d -Amino Acid Amidohydrolases Using Artificial Amidophenol Substrates and Microbial Biosensors
An, Jung-Ung,Kim, Haseong,Kwon, Kil Koang,Lee, Dae-Hee,Lee, Hyewon,Lee, Jin-Young,Lee, Seung-Goo,Park, Sung Hyun,Rha, Eugene,Yeom, Soo-Jin
, p. 1203 - 1211 (2022/01/27)
Enantiomerically pure d-amino acids are important intermediates as chiral building blocks for peptidomimetics and semisynthetic antibiotics. Here, a transcriptional factor-based screening strategy was used for the rapid screening of d-stereospecific amino acid amidase via an enzyme-specific amidophenol substrate. We used a d-threonine amidophenyl derivative to produce 2-aminophenol that serves as a putative enzyme indicator in the presence of d-threonine amidases. Comparative analyses of known bacterial species indicated that several Bacillus strains produce amidase and form putative indicators in culture media. The estimated amidase was cloned and subjected to rapid directed evolution through biosensor cells. Consequently, we characterized the F119A mutation that significantly improved the catalytic activity toward d-alanine, d-threonine, and d-glutamate. Its beneficial effects were confirmed by higher conversions and recurrent applications of the mutant enzyme, compared to the wild-type. This study showed that rapid directed evolution with biosensors coupled to designed substrates is useful to develop biocatalytic processes.
Cβ-Selective Aldol Addition of d -Threonine Aldolase by Spatial Constraint of Aldehyde Binding
Park, Sung-Hyun,Seo, Hogyun,Seok, Jihye,Kim, Haseong,Kwon, Kil Koang,Yeom, Soo-Jin,Lee, Seung-Goo,Kim, Kyung-Jin
, p. 6892 - 6899 (2021/06/28)
d-Threonine aldolase (DTA) is a useful biocatalyst that reversibly converts glycine and aldehyde to β-hydroxy-α-d-amino acid. However, low activity and poor diastereoselectivity limit its applications. Here we report DTA from Filomicrobium marinum (FmDTA) that shows much higher activity and Cβ-stereoselectivity in d-threonine production compared with those of other known DTAs. We determine the FmDTA structure at a 2.2 ? resolution and propose a DTA catalytic mechanism with a kernel of the Lys49 inner proton sink and metal ion in the aldol reaction cycle. The enzyme is rationally engineered to have high Cβ-stereoselectivity based on spatial constraint at the anti-specific aldehyde position in the mechanism, and the rational strategy is further applied to other DTAs for syn-production. The final FmDTAG179A/S312A variant exhibits a near-perfect 99.5% de value for d-threonine and maintains the de value above 93% even under kinetically unfavorable conditions. This study demonstrates how a detailed understanding of the reaction mechanism can be used for rational protein engineering.
Noncovalently Functionalized Commodity Polymers as Tailor-Made Additives for Stereoselective Crystallization
Wan, Xinhua,Wang, Zhaoxu,Ye, Xichong,Zhang, Jie
supporting information, p. 20243 - 20248 (2021/08/09)
Stereoselective inhibition of the nucleation and crystal growth of one enantiomer aided by “tailor-made” polymeric additives is an efficient method to obtain enantiopure compounds. However, the conventional preparation of polymeric additives from chiral monomers are laborious and limited in structures, which impedes their rapid optimization and applicability. Herein, we report a “plug-and-play” strategy to facilitate synthesis by using commercially available achiral polymers as the platform to attach various chiral small molecules as the recognition side-chains through non-covalent interactions. A library of supramolecular polymers made up of two vinyl polymers and six small molecules were applied with seeds in the selective crystallization of seven racemates in different solvents. They showed good to excellent stereoselectivity in yielding crystals with high enantiomeric purities in conglomerates and racemic compound forming systems. This convenient, low-cost modular synthesis strategy of polymeric additives will allow for high-efficient, economical resolution of various racemates on different scales.
Mechanism of eukaryotic serine racemase-catalyzed serine dehydration
Goto, Masaru,Hemmi, Hisashi,Ito, Tomokazu,Matsuoka, Mai,Matsushita, Kazuma,Mizobuchi, Taichi,Nasu, Ryoma,Watanabe, Soichiro,Yoshimura, Tohru
, (2020/06/08)
Eukaryotic serine racemase (SR) is a pyridoxal 5′-phosphate enzyme belonging to the Fold-type II group, which catalyzes serine racemization and is responsible for the synthesis of D-Ser, a co-agonist of the N-methyl-D-aspartate receptor. In addition to racemization, SR catalyzes the dehydration of D- and L-Ser to pyruvate and ammonia. The bifuctionality of SR is thought to be important for D-Ser homeostasis. SR catalyzes the racemization of D- and L-Ser with almost the same efficiency. In contrast, the rate of L-Ser dehydration catalyzed by SR is much higher than that of D-Ser dehydration. This has caused the argument that SR does not catalyze the direct D-Ser dehydration and that D-Ser is first converted to L-Ser, then dehydrated. In this study, we investigated the substrate and solvent isotope effect of dehydration of D- and L-Ser catalyzed by SR from Dictyostelium discoideum (DdSR) and demonstrated that the enzyme catalyzes direct D-Ser dehydration. Kinetic studies of dehydration of four Thr isomers catalyzed by D. discoideum and mouse SRs suggest that SR discriminates the substrate configuration at C3 but not at C2. This is probably the reason for the difference in efficiency between L- and D-Ser dehydration catalyzed by SR.
Trading N and O. Part 4: Asymmetric synthesis of syn-β-substituted-α-amino acids
Davies, Stephen G.,Fletcher, Ai M.,Greenaway, Catherine J.,Kennedy, Matthew S.,Mayer, Christoph,Roberts, Paul M.,Thomson, James E.
, p. 5049 - 5061 (2018/05/08)
A total of nine enantiopure syn-β-substituted-α-amino acids have been synthesised, comprising both syn-β-hydroxy-α-amino acids and syn-β-fluoro-α-amino acids. The key step in the synthetic strategy towards these syn-β-substituted-α-amino acids involves a stereospecific rearrangement, which proceeds via the intermediacy of the corresponding aziridinium ions. The requisite enantiopure syn-α-hydroxy-β-amino esters were prepared via asymmetric aminohydroxylation of the corresponding α,β-unsaturated esters followed by epimerisation of the resultant anti-α-hydroxy-β-amino esters at the C(2)-position. Subsequent activation of the α-hydroxy moiety as a leaving group followed by displacement by the β-amino substituent gave the corresponding aziridinium species. Regioselective in situ ring-opening of the aziridinium intermediates with either water or fluoride gave the corresponding syn-β-hydroxy-α-amino ester or syn-β-fluoro-α-amino ester, respectively, and N-deprotection and ester hydrolysis afforded the target syn-β-substituted-α-amino acids as single diastereoisomers in good overall yield.
Covalent Organic Frameworks with Chirality Enriched by Biomolecules for Efficient Chiral Separation
Zhang, Sainan,Zheng, Yunlong,An, Hongde,Aguila, Briana,Yang, Cheng-Xiong,Dong, Yueyue,Xie, Wei,Cheng, Peng,Zhang, Zhenjie,Chen, Yao,Ma, Shengqian
supporting information, p. 16754 - 16759 (2018/11/27)
The separation of racemic compounds is important in many fields, such as pharmacology and biology. Taking advantage of the intrinsically strong chiral environment and specific interactions featured by biomolecules, here we contribute a general strategy is developed to enrich chirality into covalent organic frameworks (COFs) by covalently immobilizing a series of biomolecules (amino acids, peptides, enzymes) into achiral COFs. Inheriting the strong chirality and specific interactions from the immobilized biomolecules, the afforded biomolecules?COFs serve as versatile and highly efficient chiral stationary phases towards various racemates in both normal and reverse phase of high-performance liquid chromatography (HPLC). The different interactions between enzyme secondary structure and racemates were revealed by surface-enhanced Raman scattering studies, accounting for the observed chiral separation capacity of enzymes?COFs.
Preparation and purification method of amino acid compound
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Paragraph 0066; 0067, (2018/06/21)
The invention relates to the field of industrial organic synthesis, in particular to a preparation and purification method of an amino acid compound. The method comprises the following steps that (1)alpha-amino nitrile compounds or hydantoin compounds or mixtures thereof are heated to react to obtain alpha-amino acid salt under the condition that volatile alkali and a suitable solvent exist; (2)after the alpha-amino acid salt obtained in step (1) is distilled, the alpha-amino acid salt is recrystallized in an organic solvent to obtain the alpha-amino acid compound. According to the method, reaction conditions are mild, materials can be recycled, and introduction of metal ions and use of ammonium carbonate salt are avoided, so that post-treatment is simple and no waste salt is generated.
A α - amino acid compound synthesis and purification method
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Paragraph 0069; 0070, (2018/05/16)
The invention relates to a synthesis and purification method for an alpha-amino acid compound. The synthesis and purification method is characterized by comprising the following steps: (1) adding substituted alpha-amino nitrile or a substituted hydantoin-based compound into alkali M(OH)x or metal oxide MxO, adding water or an alcohol and water mixed solvent, and heating for reaction to obtain alpha-amino acid salt; (2) adding ammonium carbonate or ammonium bicarbonate or introducing carbon dioxide into the solution in the step (1), separating to obtain filter liquor and precipitates MxHyCO3, performing reduced pressure concentration on the filter liquor, and recrystallizing in an alcohol solvent to obtain the alpha-amino acid compound (I). The synthesis and purification method for the alpha-amino acid compound is simple, the yield and purity of the obtained alpha-amino acid compound are high; furthermore, recycling utilization and cleaning production of materials can be realized; the synthesis and purification method is especially suitable for synthesis of the alpha-amino acid compound with high water solubility.
Cloning and characterization of D-threonine aldolase from the green alga Chlamydomonas reinhardtii
Hirato, Yuki,Tokuhisa, Mayumi,Tanigawa, Minoru,Ashida, Hiroyuki,Tanaka, Hiroyuki,Nishimura, Katsushi
, p. 18 - 23 (2017/02/05)
D-Threonine aldolase (DTA) catalyzes the pyridoxal 5’-phosphate (PLP)-dependent interconversion of D-threonine and glycine plus acetaldehyde. The enzyme is a powerful tool for the stereospecific synthesis of various β-hydroxy amino acids in synthetic organic chemistry. In this study, DTA from the green alga Chlamydomonas reinhardtii was discovered and characterized, representing the first report to describe the existence of eukaryotic DTA. DTA was overexpressed in recombinant Escherichia coli BL21 (DE3) cells; the specific activity of the enzyme in the cell-free extract was 0.8 U/mg. The recombinant enzyme was purified to homogeneity by ammonium sulfate fractionation, DEAE-Sepharose, and Mono Q column chromatographies (purified enzyme 7.0 U/mg). For the cleavage reaction, the optimal temperature and pH were 70?°C and pH 8.4, respectively. The enzyme demonstrated 90% of residual activity at 50?°C for 1?h. The enzyme catalyzed the synthesis of D- and D-allo threonine from a mixture of glycine and acetaldehyde (the diastereomer excess of D-threonine was 18%). DTA was activated by several divalent metal ions, including manganese, and was inhibited by PLP enzyme inhibitors and metalloenzyme inhibitors.
A new d-threonine aldolase as a promising biocatalyst for highly stereoselective preparation of chiral aromatic β-hydroxy-α-amino acids
Chen, Qijia,Chen, Xi,Cui, Yunfeng,Ren, Jie,Lu, Wei,Feng, Jinhui,Wu, Qiaqing,Zhu, Dunming
, p. 5964 - 5973 (2017/12/26)
d-Threonine aldolase is an enzyme belonging to the glycine-dependent aldolases, and it catalyzes the reversible aldol reaction of glycine and acetaldehyde to give d-threonine and/or d-allo-threonine. In this study, a putative d-threonine aldolase gene from Delftia sp. RIT313 was cloned and expressed in Escherichia coli BL21 (DE3). The purified enzyme (DrDTA, 47 KDa) exhibited 21.3 U mg-1 activity for the aldol addition of glycine and acetaldehyde in MES-NaOH buffer (pH 6.0) at 50 °C. Both pyridoxal 5′-phosphate and metal ions were needed for the reaction, and the existence of the metal ions enhanced the stability of the enzyme. It was found that the conversion and Cβ-stereoselectivity were dramatically influenced by the reaction temperature, co-solvent, amount of enzyme and reaction time, and it is possible to enable the reaction under kinetic control to retain suitable conversion and high stereoselectivity at the β-carbon, thus tackling the "Cβ-stereoselectivity problem". DrDTA showed high activity toward aromatic aldehydes with electron-withdrawing substituents. Under the optimized reaction conditions, phenylserines with a 2′-fluoro- or 3′-nitro-substituent were obtained with >90% conversion and >90% de. In addition, dl-threo-phenylserine and dl-threo-4-(methylsulfonyl)phenylserine were efficiently resolved with an excellent enantiomeric excess value (ee, >99%) using a whole cell biocatalyst in a two-phase system at 1.0 M and 0.3 M, respectively, the highest substrate concentration reported so far. These results suggested that DrDTA might be a promising biocatalyst for producing chiral aromatic β-hydroxy-α-amino acids.
