Construction of a Highly Diastereoselective Aldol Reaction System with <scp>l</scp>-Threonine Aldolase by Computer-Assisted Rational Molecular Modification and Medium Engineering

Zheng, Wenlong; Chen, Kaitong; Wang, Zhe; Cheng, Xu; Xu, Gang; Yang, Lirong; Wu, Jianping

doi:10.1021/acs.orglett.0c01792

Cited by 32 publications

(53 citation statements)

References 25 publications

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“…l -Threonine aldolase (LTA) is a 5′-phosphate (PLP)-dependent aldolase that catalyzes the formation of β-hydroxy-α-amino acids, important chiral building blocks for pharmaceuticals such as antibiotics and proteasome inhibitors, from glycine (Gly) and aromatic or aliphatic aldehydes (Scheme ). − The process is attractive for producing chiral products with two chiral centers (C α and C β ) in one step. − However, although LTA has a strict selectivity for C α , the selectivity for C β is moderate, hampering its wide applications in stereoselective synthesis of carbon–carbon bonds. ,− LTA has been engineered for improved C β stereoselectivity in several studies but with limited success, mainly due to an unclear mechanism determining diastereoselectivity and the lack of an appropriate directed evolution strategy. ,− Salvo et al crystallized LTA from Escherichia coli and took rational design to improve diastereoselectivity . Based on the result of docking threonine to the LTA, several amino acid residues located in the active center were mutated to other polar or nonpolar amino acid residues.…”

Section: Introductionmentioning

confidence: 99%

“…Enhancing the syn path and blocking the anti path could potentially increase the de value of the l - syn product. This hypothesis has been successfully used to guide the rational design of improved variants of LTA from Bacillus nealsonii ( Bn LTA, GenBank accession code WP_016204489.1) . Despite this great achievement, there is still a large space for the improvement of C β selectivity and it might not be fulfilled by solely the rational design method.…”

Section: Introductionmentioning

confidence: 99%

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Directed Evolution of l-Threonine Aldolase for the Diastereoselective Synthesis of β-Hydroxy-α-amino Acids

Zheng

Fang

et al. 2021

ACS Catal.

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l-Threonine aldolase (LTA) is an attractive tool in organic chemistry for catalyzing the formation of β-hydroxy-α-amino acids with two chiral centers. The enzyme has a strict selectivity for Cα of β-hydroxy-α-amino acids but a moderate selectivity for Cβ , limiting its wide applications in stereospecific carbon–carbon bond synthesis. Here, a combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy was applied to accelerate directed evolution of LTA in diastereoselectivity. A total of 27 amino acid residues lining the substrate pocket were selected and divided into two groups based on their functional region. In silico screening and site-directed saturation mutagenesis identified six (3 + 3) amino acid residues of them with a significant effect on diastereoselectivity. The ISM strategy was then performed in and between each group to obtain the best combinatorial mutation. As a result, a variant RS1 (Y8H/Y31H/I143R/N305R) was obtained with a dramatically improved preference for the synthesis of l-syn-3-[4-(methylsulfonyl)phenylserine]. The product with a de value of 99.5% (73.2% conv) was produced in a 20 L reactor, which is promising in the industrial synthesis of aromatic l-syn-β-hydroxy-α-amino acids with LTA. The variant also represented a significant selective improvement to other l-phenylserine derivatives. The de values of 2-NO2-, 4-NO2-, H-, and 4-CH4-substituted l-phenylserine derivatives were more than 99% syn by dynamic control. The insight of the mutant model suggests that the binding pocket of the active center was reshaped.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Directed Evolution of l-Threonine Aldolase for the Diastereoselective Synthesis of β-Hydroxy-α-amino Acids

Zheng

Fang

et al. 2021

ACS Catal.

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“…Moreover, other para -substituted benzaldehydes with both electron-donating and electron-withdrawing groups were well tolerated by the cascade reaction combining Ec TK1_YYF and ATA117_ACHH, enabling the highly diastereo- and enantioselective (95–97% de and >99% ee) synthesis of the corresponding aminodiol products with moderate to good yields (51–72%) within 6 h (Table , entries 3–7 and Figures S15 and S17–S28). Notably, the para -substituted phenylserinols with fluorine, chlorine, and bromine groups furnished in our cascade reaction possessed higher conversion and higher de values than the reported corresponding para -substituted intermediates of florfenicol, ,, which suggested that our cascade route exhibited a broad substrate scope.…”

Section: Resultsmentioning

confidence: 63%

“…The enzyme cascade combining EcTK1_YYH and ATA117_ACHH in sequential mode for the synthesis of (1R,2R)-2-amino-1-(4- . Notably, the para-substituted phenylserinols with fluorine, chlorine, and bromine groups furnished in our cascade reaction possessed higher conversion and higher de values than the reported corresponding para-substituted intermediates of florfenicol, 24,26,63 which suggested that our cascade route exhibited a broad substrate scope. Although benzaldehyde derivatives bear different parasubstitutions, they gave all products with absolute enantioselectivity (>99% ee) (Tables 1 and 2), which suggested that the evolved mutations (V69A/F122C/I157H/F225H) of ATA117 responsible for manipulating the enantiopreference toward the chiral hydroxyketone do not impair the innate (R)-stereospecificity of the enzyme.…”

Section: ■ Results and Discussionmentioning

confidence: 65%

One-Pot Asymmetric Synthesis of an Aminodiol Intermediate of Florfenicol Using Engineered Transketolase and Transaminase

et al. 2021

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Florfenicol is the 3′-fluoro derivative of thiamphenicol and has been widely used in veterinary medicine for its high antibacterial activity and safety. However, the development of simplified and environmentally friendly catalytic methods for the stereoselective production of florfenicol is a key challenge. Herein, we established a highly stereoselective enzymatic one-pot reaction for the synthesis of an aminodiol intermediate of florfenicol bearing two stereocenters from industrial raw material 4-(methylsulfonyl) benzaldehyde by coupling transketolase (TK) and ω-transaminase (TA). The enantioselectivity of TK from E. coli was converted from (S) (93% ee) to (R) (95% ee), and we also inverted the enantiopreference (E(S) = 9 to E(R) = 12) and ketone/ aldehyde substrate selectivity of TA ATA117 via structure-guided enzyme engineering. Docking calculations and molecular dynamics simulations of the wild-type and mutant enzymes unveiled the molecular basis for enzymatic stereocontrol. Using the engineered TK and TA, (1R,2R)-p-methylsulfonyl phenylserinol was biosynthesized with good yield (76%) and high stereoselectivity (96% de and >99% ee). Our work established an enzymatic synthetic route to (1R,2R)-p-methylsulfonyl phenylserinol, facilitating the development of a chemoenzymatic method for producing florfenicol.

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“…β-Hydroxy-α-amino acids and their fluorinated derivatives are key intermediates for many drugs, which were used for the treatment of Parkinson’s disease immunodeficiency and inflammation (Rocha et al 2019 ; Scott et al 2017 ). Pyridoxal 5´-phosphate (PLP)-dependent aldolases are regarded as the key biocatalysts for the formation of C–C bonds and have become powerful tools for the sustainable synthesis of complex molecules, especially fluorinated β-hydroxy-α-amino acids through aldol condensation (Chen et al 2019a ; Fang et al 2019 ; Zheng et al 2020 , 2021 ). The formation of two chiral stereocenters is the main feature of this reaction.…”

Section: Enzymatic Synthesis Of Fluorinated Compoundsmentioning

confidence: 99%

Enzymatic synthesis of fluorinated compounds

Cheng

2021

Appl Microbiol Biotechnol

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Fluorinated compounds are widely used in the fields of molecular imaging, pharmaceuticals, and materials. Fluorinated natural products in nature are rare, and the introduction of fluorine atoms into organic compound molecules can give these compounds new functions and make them have better performance. Therefore, the synthesis of fluorides has attracted more and more attention from biologists and chemists. Even so, achieving selective fluorination is still a huge challenge under mild conditions. In this review, the research progress of enzymatic synthesis of fluorinated compounds is summarized since 2015, including cytochrome P450 enzymes, aldolases, fluoroacetyl coenzyme A thioesterases, lipases, transaminases, reductive aminases, purine nucleoside phosphorylases, polyketide synthases, fluoroacetate dehalogenases, tyrosine phenol-lyases, glycosidases, fluorinases, and multienzyme system. Of all enzyme-catalyzed synthesis methods, the direct formation of the C-F bond by fluorinase is the most effective and promising method. The structure and catalytic mechanism of fluorinase are introduced to understand fluorobiochemistry. Furthermore, the distribution, applications, and future development trends of fluorinated compounds are also outlined. Hopefully, this review will help researchers to understand the significance of enzymatic methods for the synthesis of fluorinated compounds and find or create excellent fluoride synthase in future research. Key points • Fluorinated compounds are distributed in plants and microorganisms, and are used in imaging, medicine, materials science . • Enzyme catalysis is essential for the synthesis of fluorinated compounds . • The loop structure of fluorinase is the key to forming the C-F bond . Supplementary Information The online version contains supplementary material available at 10.1007/s00253-021-11608-0.

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Construction of a Highly Diastereoselective Aldol Reaction System with l-Threonine Aldolase by Computer-Assisted Rational Molecular Modification and Medium Engineering

Cited by 32 publications

References 25 publications

Directed Evolution of l-Threonine Aldolase for the Diastereoselective Synthesis of β-Hydroxy-α-amino Acids

Directed Evolution of l-Threonine Aldolase for the Diastereoselective Synthesis of β-Hydroxy-α-amino Acids

One-Pot Asymmetric Synthesis of an Aminodiol Intermediate of Florfenicol Using Engineered Transketolase and Transaminase

Enzymatic synthesis of fluorinated compounds

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