Synthesis Of Chiral Amino Acids Research Articles

Styrene Oxide Isomerase-Catalyzed Meinwald Rearrangement in Cascade Biotransformations: Synthesis of Chiral and/or Natural Chemicals.

Styrene oxide isomerase (SOI) catalyzes the Meinwald rearrangement of aryl epoxides to carbonyl compounds via a 1,2-trans-shift in a stereospecific manner. A number of cascade biotransformations with SOI-catalyzed epoxide isomerization as a key step have been developed to convert readily available substrates into valuable chiral chemicals. Cascade conversion of terminal or internal alkenes into chiral acids, alcohols or amines was achieved, which involved SOI-catalyzed enantio-retentive isomerization of terminal epoxides via 1,2-H shift, or internal epoxides via 1,2-methyl shift. SOI-involved cascades were also developed to convert racemic epoxides into chiral acids or amines via dynamic kinetic resolution. Additionally, combining SOI-catalyzed isomerization with enantioselective C-C bond forming enzymes enabled the synthesis of chiral amino acids or amino alcohols from racemic epoxides. Finally, integration of SOI-involved cascades with biosynthesis pathways allowed for the direct utilization of renewable substrates for the sustainable synthesis of high-value natural chemicals such as alcohols, acids, and esters.

Rational identification of a high catalytic efficiency leucine dehydrogenase and process development for efficient synthesis of l-phenylglycine.

Enzymatic asymmetric synthesis of chiral amino acids has great industrial potential. However, the low catalytic efficiency of high-concentration substrates limits their industrial application. Herein, using a combination of substrate catalytic efficiency prediction based on "open to closed" conformational change and substrate specificity prediction, a novel leucine dehydrogenase (TsLeuDH), with high substrate catalytic efficiency toward benzoylformic acid (BFA) for producing l-phenylglycine (l-Phg), was directly identified from 4695 putative leucine dehydrogenases in a public database. The specific activity of TsLeuDH was determined to be as high as 4253.8Umg-1 . Through reaction process optimization, a high-concentration substrate (0.7m) was efficiently and completely converted within 90min in a single batch, without any external coenzyme addition. Moreover, a continuous flow-feeding approach was designed using gradient control of the feed rate to reduce substrate accumulation. Finally, the highest overall substrate concentration of up to 1.2m BFA could be aminated to l-Phg with conversion of >99% in 3h, demonstrating that this new combination of enzyme process development is promising for large-scale application of l-Phg.

Asymmetric Synthesis of Enantiomerically Pure Aliphatic and Aromatic D-Amino Acids Catalyzed by Transaminase from Haliscomenobacter hydrossis

D-amino acids are valuable building blocks for the synthesis of biologically active compounds and pharmaceuticals. The asymmetric synthesis of chiral amino acids from prochiral ketones using stereoselective enzymes is a well-known but far from exhausted approach for large-scale production. Herein, we investigated a pyridoxal-5′-phosphate-dependent D-amino acid transaminase from Haliscomenobacter hydrossis as a potential biocatalyst for the enzymatic asymmetric synthesis of optically pure aliphatic and aromatic D-amino acids. We studied the catalytic efficiency and stereoselectivity of transaminase from H. hydrossis in the amination of aliphatic and aromatic α-keto acids, using D-glutamate as a source of the amino group. We constructed a one-pot three-enzyme system, which included transaminase and two auxiliary enzymes, hydroxyglutarate dehydrogenase, and glucose dehydrogenase, to produce D-amino acids with a product yield of 95–99% and an enantiomeric excess of more than 99%. We estimated the stability of the transaminase and the cofactor leakage under reaction conditions. It was found that a high concentration of α-keto acids as well as a low reaction temperature (30 °C) can reduce the cofactor leakage under reaction conditions. The obtained results demonstrated the efficiency of transaminase from H. hydrossis in the asymmetric synthesis of enantiomerically pure D-amino acids.

Development of an NAD(H)‐Driven Biocatalytic System for Asymmetric Synthesis of Chiral Amino Acids

AbstractChiral amino acids are extensively applied in the pharmaceutical, food, cosmetic, and agricultural industries. As a representative example, l‐phosphinothricin (l‐PPT, a chiral non‐natural amino acid) is a broad‐spectrum herbicide. An NAD(H)‐driven biocatalytic system for the asymmetric synthesis of chiral amino acids (focused on l‐PPT) with high efficiency and low cost is highly desired. The key for the development of such biocatalytic system is to obtain an NADH‐dependent biocatalyst with high catalytic performance toward l‐PPT pro‐ketone PPO. Herein, an engineered glutamate dehydrogenase from Lysinibacillus composti (LcGluDH) with desired activity was obtained by gene mining and protein engineering. In silico analyses suggested that the volume of substrate‐binding pocket was substantially enlarged from 330.5 Å3 to 409.6 Å3. The stability of LcGluDH was increased (Tm value increased from 47.3 °C to 65.3 °C) by introducing positively charged amino acids or aromatic amino acids at position 375. The desired biocatalytic system was constructed by coupling the engineered LcGluDH and an NAD+‐dependent FDH. Through this biocatalytic system, the batch production of l‐PPT exhibited high space‐time yield (207.3 g ⋅ L−1 ⋅ day−1) with strict stereoselectivity (ee of l‐PPT>99%). Furthermore, eight other chiral amino acids were synthesised by the developed NAD(H)‐driven biocatalytic system with high ee values.magnified image

Identification and Rational Engineering of a High Substrate‐Tolerant Leucine Dehydrogenase Effective for the Synthesis of L‐tert‐Leucine

AbstractThe asymmetric synthesis of chiral amino acids by leucine dehydrogenases has great potential for industrialization; however, the inhibitory effect of high‐concentration substrates limits its large‐scale application. Herein, using structure‐guided genome mining based on sequence‐structure prediction of substrate tolerance and specificity, a novel leucine dehydrogenase (LaLeuDH) from Labrenzia aggregate was identified and characterized, which exhibited the highest substrate tolerance and excellent activity to trimethyl pyruvate, even at 1.5 M concentration. Moreover, based on coenzyme binding structural information and sequence alignment, directed evolution of LaLeuDH was performed to increase affinity for NADH. The obtained variant D153 N/H191 N resulted in 25‐fold improved affinity for NADH, with 50‐fold enhanced catalytic efficiency (kcat/Km) of 40464.6 mM−1 s−1. Finally, through a combination of the above two strategies, as high as 1.5 M substrate could be completely converted in 18 h without coenzyme addition, demonstrating that this engineered enzyme has promising prospects for industrialization.

Crystal Structures and Catalytic Mechanism of l-erythro-3,5-Diaminohexanoate Dehydrogenase and Rational Engineering for Asymmetric Synthesis of β-Amino Acids.

Amino acid dehydrogenases (AADHs) have shown considerable potential as biocatalysts in the asymmetric synthesis of chiral amino acids. However, compared to the widely studied α-AADHs, limited knowledge is available about β-AADHs that enable the synthesis of β-amino acids. Herein, we report the crystal structures of a l-erythro-3,5-diaminohexanoate dehydrogenase and its variants, the only known member of β-AADH family. Crystal structure analysis, site-directed mutagenesis studies and quantum chemical calculations revealed the differences in the substrate binding and catalytic mechanism from α-AADHs. A number of rationally engineered variants were then obtained with improved activity (by 110-800 times) toward various aliphatic β-amino acids without an enantioselectivity trade-off. Two β-amino acids were prepared by using the outstanding variants with excellent enantioselectivity (>99 % ee) and high isolated yields (86-87 %). These results provide important insights into the molecular mechanism of 3,5-DAHDH, and establish a solid foundation for further design of β-AADHs for the asymmetric synthesis of β-amino acids.

Crystal Structures and Catalytic Mechanism of l‐erythro‐3,5‐Diaminohexanoate Dehydrogenase and Rational Engineering for Asymmetric Synthesis of β‐Amino Acids

AbstractAmino acid dehydrogenases (AADHs) have shown considerable potential as biocatalysts in the asymmetric synthesis of chiral amino acids. However, compared to the widely studied α‐AADHs, limited knowledge is available about β‐AADHs that enable the synthesis of β‐amino acids. Herein, we report the crystal structures of a l‐erythro‐3,5‐diaminohexanoate dehydrogenase and its variants, the only known member of β‐AADH family. Crystal structure analysis, site‐directed mutagenesis studies and quantum chemical calculations revealed the differences in the substrate binding and catalytic mechanism from α‐AADHs. A number of rationally engineered variants were then obtained with improved activity (by 110–800 times) toward various aliphatic β‐amino acids without an enantioselectivity trade‐off. Two β‐amino acids were prepared by using the outstanding variants with excellent enantioselectivity (>99 % ee) and high isolated yields (86–87 %). These results provide important insights into the molecular mechanism of 3,5‐DAHDH, and establish a solid foundation for further design of β‐AADHs for the asymmetric synthesis of β‐amino acids.

Construction of a Self-Purification and Self-Assembly Coenzyme Regeneration System for the Synthesis of Chiral Drug Intermediates.

As one of the important research contents of synthetic biology, the construction of a regulatory system exhibits great potential in the synthesis of high value-added chemicals such as drug intermediates. In this work, a self-assembly coenzyme regeneration system, leucine dehydrogenase (LeuDH)–formate dehydrogenase (FDH) protein co-assembly system, was constructed by using the polypeptide, SpyTag/SpyCatcher. Then, it was demonstrated that the nonchromatographic inverse transition cycling purification method could purify intracellular coupling proteins and extracellular coupling proteins well. The conversion rate of the pure LeuDH–FDH protein assembly (FR-LR) was shown to be 1.6-fold and 32.3-fold higher than that of the free LeuDH–FDH system (LeuDH + FDH) and free LeuDH, respectively. This work has paved a new way of constructing a protein self-assembly system and engineering self-purification coenzyme regeneration system for the synthesis of chiral amino acids or chiral α-hydroxy acids.

Catalytic enantioselective synthesis of β-amino alcohols by nitrene insertion

Chiral β-amino alcohols are important building blocks for the synthesis of drugs, natural products, chiral auxiliaries, chiral ligands and chiral organocatalysts. The catalytic asymmetric β-amination of alcohols offers a direct strategy to access this class of molecules. Herein, we report a general intramolecular C(sp3)–H nitrene insertion method for the synthesis of chiral oxazolidin-2-ones as precursors of chiral β-amino alcohols. Specifically, the ring-closing C(sp3)–H amination of N -benzoyloxycarbamates with 2 mol% of a chiral ruthenium catalyst provides cyclic carbamates in up to 99% yield and with up to 99% ee. The method is applicable to benzylic, allylic, and propargylic C–H bonds and can even be applied to completely non-activated C(sp3)–H bonds, although with somewhat reduced yields and stereoselectivities. The obtained cyclic carbamates can subsequently be hydrolyzed to obtain chiral β-amino alcohols. The method is very practical as the catalyst can be easily synthesized on a gram scale and can be recycled after the reaction for further use. The synthetic value of the new method is demonstrated with the asymmetric synthesis of a chiral oxazolidin-2-one as intermediate for the synthesis of the natural product aurantioclavine and chiral β-amino alcohols that are intermediates for the synthesis of chiral amino acids, indane-derived chiral Box-ligands, and the natural products dihydrohamacanthin A and dragmacidin A.

Artificial Biocatalytic Cascade with Three Enzymes in One Pot for Asymmetric Synthesis of Chiral Unnatural Amino Acids

Two biocatalytic reactions, transamination catalyzed by transaminases and reductive amination catalyzed by amino acid dehydrogenases, can be used for asymmetric synthesis of optically pure unnatural amino acids. However, although transaminases show a great diversity and broad substrate spectrum, most transaminase reactions are reversible, while amino acid dehydrogenases catalyze reductive amination irreversibly but with strict substrate specificity. Accordingly, herein we developed a tri‐enzyme one‐pot reaction system to exploit the respective advantages of transaminases and amino acid dehydrogenases, while overcoming the disadvantages of each. In this work, representatives of all four subgroups of transaminases coupled with different amino acid dehydrogenases to produce five l‐ and four d‐ unnatural amino acid products, using ammonia and the co‐enzyme NAD(P)H, which is regenerated by a robust alcohol dehydrogenase with 2‐propanol as cheap cosubstrate. The complete conversion and high enantiopurity (ee > 99 %) of the products, demonstrated it as an ideal alternative for asymmetric synthesis of chiral amino acid compounds.

Improving Catalytic Efficiency and Changing Substrate Spectrum for Asymmetric Biocatalytic Reductive Amination.

With the advantages of biocatalytic method, enzymes have been excavated for the synthesis of chiral amino acids by the reductive amination of ketones, offering a promising way of producing pharmaceutical intermediates. In this work, a robust phenylalanine dehydrogenase (PheDH) with wide substrate spectrum and high catalytic efficiency was constructed through rational design and active-site-targeted, site-specific mutagenesis by using the parent enzyme from Bacillus halodurans. Active sites with bonding substrate and amino acid residues surrounding the substrate binding pocket, 49L-50G-51G, 74M,77K, 122G-123T-124D-125M, 275N, 305L and 308V of the PheDH, were identified. Noticeably, the new mutant PheDH (E113D-N276L) showed approximately 6.06-fold increment of kcat/Km in the oxidative deamination and more than 1.58-fold in the reductive amination compared to that of the wide type. Meanwhile, the PheDHs exhibit high capacity of accepting benzylic and aliphatic ketone substrates. The broad specificity, high catalytic efficiency and selectivity, along with excellent thermal stability, render these broad-spectrum enzymes ideal targets for further development with potential diagnostic reagent and pharmaceutical compounds applications.

Gas-Phase Stereoinversion in Aspartic Acid: Reaction Pathways, Computational Spectroscopic Analysis, and Its Astrophysical Relevance.

Noncatalytic reaction pathways for the gas-phase stereoinversion in aspartic acid are mapped employing a global reaction route mapping strategy using quantum mechanical computations. The species including the transition states (TSs) traced along the stereoinversion pathways are characterized using rotational and vibrational computational spectroscopic analysis while accounting for the vibrational corrections to rotational constants and anharmonic effects. Notably, the TS structures traced along the stereochemical pathways resemble the achiral ammonium ylide and imine intermediates as observed in the Strecker synthesis of chiral amino acids. A few of the probable stereoinversion pathways proposed proceed through the proton or hydrogen atom transfer. The feasibility of the pathways under conditions akin to interstellar medium (ISM) is further discussed in terms of natural bond orbital analysis. The stereoinversion pathways proposed in this work may proceed via photoirradiation in the ISM, which though can be revealed by exploring the excited-state potential energy surface. In this context, the spectroscopic data generated in this work can provide valuable assistance toward the astrophysical detection of chiral molecules in outer space.

Enzymatic asymmetric synthesis of chiral amino acids.

Chiral amino acids are extensively applied in the pharmaceutical, food, cosmetic, agricultural, and feedstuff industries. The development of synthetic methodologies for optically pure amino acids has been driven by their significant applications. Among the various synthesis methods for the production of chiral amino acids, enzymatic asymmetric synthesis is a unique preparation strategy that shows great potential. This review provides an overview of the reported methods for enzymatic asymmetric synthesis of chiral amino acids, including asymmetric reductive amination of keto acids, asymmetric transfer of an amino group to keto acids, enantioselective addition of ammonia to α,β-unsaturated acids, and aldol condensation of an amino acid to aldehydes.

Asymmetric Amination of 3‐(2‐Oxoindolin‐3‐ylidene)butanoates Catalyzed by a Cinchona‐Derived Alkaloid

AbstractHere we report the asymmetric amination of 3‐(2‐oxoindolin‐3‐ylidene)butanoates catalyzed by a cinchona‐derived alkaloid, yielding products with an N‐containing stereocenter in high yields and moderate enantioselectivity. The developed process could be an attractive approach for the synthesis of chiral amino acids.

Continuous colorimetric assay that enables high-throughput screening of N-acetylamino acid racemases.

N-Acetyl amino acid racemases (NAAARs) have demonstrated their potential in the enzymatic synthesis of chiral amino acids, molecules of significant biotechnology interest. In order to identify novel activities and to improve these enzymes by engineering approaches, suitable screening methods are necessary. Previous engineering of the NAAAR from Amycolatopsis Ts-1-60 was achieved by relying on an in vivo selection system that linked the viability of an E. coli L-methionine auxotroph to the activity of the improved enzyme. However, this assay was only suitable for the screening of N-acetyl-D-methionine, therefore limiting the potential to evolve this enzyme toward other natural or non-natural acetylated amino acids. Here, we report the optimization and application of a spectrophotometric microtiter-plate-based assay for NAAAR. The assay is based on the detection of the amino acid reaction product formed by hydrolysis of the N-acylated substrate by an L-amino acid acylase and its subsequent oxidation by an FAD-dependent L-amino acid oxidase (L-AAO). Cofactor recycling of the L-AAO leads to the formation of hydrogen peroxide which is easily monitored using horseradish peroxidase (HRP) and o-dianisidine. This method allowed for the determination of the kinetic parameters of NAAAR and led to the identification of N-acetyl-D-naphthylalanine as a novel NAAAR substrate. This robust method is also suitable for the high-throughput screening of NAAAR mutant gene libraries directly from cell lysates.

The first example of Ni(II)-catalyzed asymmetric addition of β-oxophosphonate to nitroalkene

Polyfunctional chiral phosphonates are of interest as intermediates in the synthesis of bioactive molecules [1–5]. A possible approach to preparation of these compounds is asymmetric addition of β-oxophosphonates to nitroalkenes [6]. Earlier, we have shown that complexes of nickel [7], cobalt, and manganese [8] with chiral diamines are efficient catalysts for enantioselective addition of 1,3dicarbonyl compounds to nitroalkenes. This reaction has been used as a key step in synthesis of chiral amino acids showing neurotropic activity [9], and of alicyclic polyfunctional derivatives [10].

New heterogeneous catalysts for the synthesis of chiral amino acids: Functionalization of organic resins with chiral salen complexes

Four new heterogeneous catalysts were synthesized by covalent attachment of vanadium(V) and aluminum(III) salen complexes to polystyrene polymers, namely, Merrifield and JandaJel resins. The solid catalysts were characterized by analytical and spectroscopic techniques and tested in the asymmetric addition of hydrogen cyanide (generated in situ from TMSCN) to N-benzyl benzylimine (Strecker type reaction). Heterogeneous vanadium(V) catalysts are more efficient than the Al(III) catalysts, as expected from comparison with the corresponding homogeneous systems. The activity of the vanadium(V) heterogeneous catalysts is similar to the homogenous counterpart (after 4h of reaction at −40°C using 10mol% of catalysts), while the enantiomeric excess was slightly inferior to the one obtained with the corresponding homogeneous catalysts. The Janda-V(V) heterogeneous catalyst can be reused by simple filtration up to three times without significative loss of conversion and enantioselectivity. This is the first study dedicated to the synthesis of asymmetric amino acids through the Strecker reaction using heterogeneous salen catalysts.

A convenient synthesis of chiral amino acid derived 3,4-dihydro-2 H-benzo[ b][1,4]thiazines and antibiotic levofloxacin

A series of 3,4-dihydro- 2H-benzo[ b][1,4]thiazine derivatives 8a– g were synthesized via- a copper-catalyzed intramolecular N-aryl amination reaction on substituted 2-(2-bromophenylthio)-ethanamines which were synthesized by the nucleophilic substitution reaction of 2-bromobenzenethiol with Boc-protected amino alcohol derivatives. This strategy provides a short and an efficient entry to ( S)-3-methyl-1,4-benzoxazine 12, an advanced synthetic intermediate for the synthesis of levofloxacin.

Development of a new class of C1-symmetric bisphosphine ligands for rhodium-catalyzed asymmetric hydrogenation

A new class of C 1-symmetric bisphosphine ligands with three hindered quadrants have been obtained through facile synthesis from chiral BINOL derivatives. Their rhodium complexes have exhibited high enantioselectivities (up to 98% ee) in the asymmetric hydrogenation of various unsaturated prochiral olefins, providing an efficient catalytic system for the enantioselective synthesis of chiral amino acids and amines.

Synthesis of a chiral amino acid with bicyclo[1.1.1]pentane moiety and its incorporation into linear and cyclic antimicrobial peptides

The synthesis of the lipophilic chiral amino acid 1 bearing the bicyclo[1.1.1]pentane moiety is described. Linear and cyclic hexapeptides of the type Arg-Arg-Xaa-Yaa-Arg-Phe containing 1 instead of one or two tryptophan residues are prepared by solid phase peptide synthesis and the antimicrobial and hemolytic activity of the peptides obtained are discussed.