Beyond the Chemical Step: The Role of Substrate Access in Acyltransferase from <i>Mycobacterium smegmatis</i>

Carvalho, Henrique F.; Mestrom, Luuk; Hanefeld, Ulf; Pleiss, Jürgen

doi:10.1021/acscatal.4c00812

Bienzymatic Dynamic Kinetic Resolution of Secondary Alcohols by Esterification/Racemization in Water

Rudzka,

Reiter,

Kroutil

et al. 2024

Angewandte Chemie

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Dynamic kinetic resolution (DKR) is a key method used to prepare optically pure compounds in 100% theoretical yield starting from racemic substrates by combining the interconversion of substrate enantiomers with an enantioselective transformation. Various chemoenzymatic DKR approaches have been developed to deracemize secondary alcohols, typically requiring an organic solvent to facilitate enantioselective acylation, primarily catalyzed by lipases, alongside racemization mediated by an achiral, non‐enzymatic catalyst. Achieving both steps in an aqueous solution remained elusive. Here, we report a DKR of racemic sec‐alcohols in an aqueous solution requiring only two biocatalysts. The first key to success was to achieve fast racemization in a buffer employing a non‐stereoselective variant of alcohol dehydrogenase (Lk‐ADH‐Prince) via a hydrogen‐borrowing oxidation‐reduction sequence. Engineered variants of the acyltransferase from Mycobacterium smegmatis (MsAcT) enabled enantioselective acyl transfer in water. Besides the appropriate choice of the enzymes, identifying a suitable acyl donor was a second key to the success. The DKR was successfully demonstrated using (R)‐selective MsAcT variants for a broad range of racemic (hetero)benzylic alcohols using 2,2,2‐trifluoroethyl acetate as the acyl donor, yielding (R)‐acetates with up to >99% conv. and high‐to‐excellent optical purity (83–99.9% ee). The (S)‐acetates were accessible using a stereocomplementary (S)‐selective MsAcT variant.

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Bienzymatic Dynamic Kinetic Resolution of Secondary Alcohols by Esterification/Racemization in Water

Rudzka,

Reiter,

Kroutil

et al. 2024

Angew Chem Int Ed

0

View full text Add to dashboard Cite

Dynamic kinetic resolution (DKR) is a key method used to prepare optically pure compounds in 100% theoretical yield starting from racemic substrates by combining the interconversion of substrate enantiomers with an enantioselective transformation. Various chemoenzymatic DKR approaches have been developed to deracemize secondary alcohols, typically requiring an organic solvent to facilitate enantioselective acylation, primarily catalyzed by lipases, alongside racemization mediated by an achiral, non‐enzymatic catalyst. Achieving both steps in an aqueous solution remained elusive. Here, we report a DKR of racemic sec‐alcohols in an aqueous solution requiring only two biocatalysts. The first key to success was to achieve fast racemization in a buffer employing a non‐stereoselective variant of alcohol dehydrogenase (Lk‐ADH‐Prince) via a hydrogen‐borrowing oxidation‐reduction sequence. Engineered variants of the acyltransferase from Mycobacterium smegmatis (MsAcT) enabled enantioselective acyl transfer in water. Besides the appropriate choice of the enzymes, identifying a suitable acyl donor was a second key to the success. The DKR was successfully demonstrated using (R)‐selective MsAcT variants for a broad range of racemic (hetero)benzylic alcohols using 2,2,2‐trifluoroethyl acetate as the acyl donor, yielding (R)‐acetates with up to >99% conv. and high‐to‐excellent optical purity (83–99.9% ee). The (S)‐acetates were accessible using a stereocomplementary (S)‐selective MsAcT variant.

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Irreversible transesterification reactions in aqueous media

Hollmann,

Wu,

Ma

et al. 2024

Preprint

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Transesterification reactions are fundamental transformations in organic chemistry. However, performing these reactions in aqueous media is challenging due to the competing hydrolysis reaction. Kinetic control and the use of activated acyl donors offer potential solutions, but they are difficult to implement. In this study, we present a mutant of alcohol oxidase from Phanerochaete chrysosporium (PcAOx-VPN) that exhibits no hydrolytic activity. Its hydrophobic active site efficiently excludes water, enabling irreversible transesterification reactions in water—previously considered impossible—thereby opening novel opportunities for chemical synthesis. Remarkably, PcAOx-VPN, as a flavin-containing enzyme, catalyzes transesterification, a promiscuous activity not previously known in this enzyme class, expanding the functional repertoire of these versatile enzymes.

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Beyond the Chemical Step: The Role of Substrate Access in Acyltransferase from Mycobacterium smegmatis

Cited by 3 publications

References 74 publications

Bienzymatic Dynamic Kinetic Resolution of Secondary Alcohols by Esterification/Racemization in Water

Bienzymatic Dynamic Kinetic Resolution of Secondary Alcohols by Esterification/Racemization in Water

Bienzymatic Dynamic Kinetic Resolution of Secondary Alcohols by Esterification/Racemization in Water

Irreversible transesterification reactions in aqueous media

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