Cloning, purification and characterization of the 6-phospho-3-hexulose isomerase YckF from Bacillus subtilis

The enzyme 6-phospho-3-hexulose isomerase (YckF) from Bacillus subtilis has been prepared and crystallized in a form suitable for X-ray crystallographic analysis. Crystals were grown by the hanging-drop method at 291 K using polyethylene glycol 2000 monomethylether as precipitant. They diffract beyond 1.7 A using an in-house Cu Kalpha source and belong to either space group P6(5)22 or P6(1)22, with unit-cell parameters a = b = 72.4, c = 241.2 A, and have two molecules of YckF in the asymmetric unit

Combining aldolases and transaminases for the synthesis of 2‑amino-4-hydroxybutanoic acid

Amino acids are of paramount importance as chiral building blocks of life, for drug development in modern medicinal chemistry, and for the manufacture of industrial products. In this work, the stereoselective synthesis of (S)- and (R)-2-amino-4-hydroxybutanoic acid was accomplished using a systems biocatalysis approach comprising a biocatalytic one-pot cyclic cascade by coupling of an aldol reaction with an ensuing stereoselective transamination. A class II pyruvate aldolase from E. coli, expressed as a soluble fusion protein, in tandem with either an S- or R-selective, pyridoxal phosphate dependent transaminase was used as a catalyst to realize the conversion, with formaldehyde and alanine being the sole starting materials. Interestingly, the class II pyruvate aldolase was found to tolerate formaldehyde concentrations of up to 1.4 M. The cascade system was found to reach product concentrations for (S)- or (R)-2-amino-4-hydroxybutanoic acid of at least 0.4 M, rendering yields between 86% and >95%, respectively, productivities of >80 g L–1 d–1, and ee values of >99%.This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 635595 (CarbaZymes), the Ministerio de Economía y Competitividad (MINECO), the Fondo Europeo de Desarrollo Regional (FEDER) (grant no. CTQ2015-63563-R to P.C.), and COST action CM1303 Systems Biocatalysis.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

Enantioselective Synthesis of Pharmaceutically Active γ-Aminobutyric Acids Using a Tailor-Made Artificial Michaelase in One-Pot Cascade Reactions

Chiral γ-aminobutyric acid (GABA) analogues represent abundantly prescribed drugs, which are broadly applied as anticonvulsants, as antidepressants, and for the treatment of neuropathic pain. Here we report a one-pot two-step biocatalytic cascade route for synthesis of the pharmaceutically relevant enantiomers of γ-nitrobutyric acids, starting from simple precursors (acetaldehyde and nitroalkenes), using a tailor-made highly enantioselective artificial “Michaelase” (4-oxalocrotonate tautomerase mutant L8Y/M45Y/F50A), an aldehyde dehydrogenase with a broad non-natural substrate scope, and a cofactor recycling system. We also report a three-step chemoenzymatic cascade route for the efficient chemical reduction of enzymatically prepared γ-nitrobutyric acids into GABA analogues in one pot, achieving high enantiopurity (e.r. up to 99:1) and high overall yields (up to 70%). This chemoenzymatic methodology offers a step-economic alternative route to important pharmaceutically active GABA analogues, and highlights the exciting opportunities available for combining chemocatalysts, natural enzymes, and designed artificial biocatalysts in multistep syntheses

Enantioselective Synthesis of Pharmaceutically Relevant Bulky Arylbutylamines Using Engineered Transaminases

ATAs engineered for having an enlarged small binding pocket were applied for the synthesis of enantiomerically pure (R)‐benzo[1,3]dioxol‐5‐yl‐butylamine, a chiral component of human leukocyte elastase inhibitor DMP 777 (L‐694,458). Kinetic resolution of the racemic amine was performed by using the L59A variant of the (S)‐selective ATA from Chromobacterium violaceum (Cv‐ATA), providing the residual (R)‐enantiomer in excellent yield and >99% ee. At moderate enzyme loading and absence of co‐solvent, high volumetric productivity of 0.22 mol L⁻¹ h⁻¹ (42.5 g L⁻¹ h⁻¹) was achieved. Complementarily, the (S)‐enantiomer was generated via kinetic resolution using the (R)‐selective ATA‐117‐Rd11 from Arthrobacter sp. with acetone as the amino acceptor. In an alternative approach, we employed ATA‐117‐Rd11 for the asymmetric amination of the prochiral ketone precursor, which at 86% conversion gave the (R)‐benzo[1,3]dioxol‐5‐yl‐butylamine with excellent >99% ee. We further evaluated the utility of Cv‐ATA L59A for the asymmetric synthesis of pharmaceutically relevant (S)‐1‐phenylbutan‐1‐amine, a chiral component of the deubiquitinase inhibitor degrasyn (WP1130). The enzyme showed good tolerance to high concentrations of isopropylamine, producing (S)‐1‐phenylbutan‐1‐amine in enantiomerically pure form (>99% ee)

An engineered cytidine deaminase for biocatalytic production of a key intermediate of the COVID-19 antiviral Molnupiravir

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Synthesis of Stereoenriched Piperidines via Chemo- Enzymatic Dearomatization of Activated Pyridines

The development of efficient and sustainable methods for the synthesis of nitrogen heterocycles is an important goal for the chemical industry. In particular, substituted chiral piperidines are prominent targets due to their prevalence in medicinally relevant compounds and their precursors. A potential biocatalytic approach to the synthesis of this privileged scaffold would be the asymmetric dearomatization of readily assembled activated pyridines. However, nature has yet to yield a suitable biocatalyst specifically for this reaction. Here, by combining chemical synthesis and biocatalysis, we present a general chemo-enzymatic approach for the asymmetric dearomatization of activated pyridines for the preparation of substituted piperidines with precise stereochemistry. The key step involves a stereoselective one-pot amine oxidase/ene imine reductase cascade to convert N-substituted tetrahydropyridines to stereo-defined 3- and 3,4-substituted piperidines. This chemo-enzymatic approach has proved useful for key transformations in the syntheses of the antipsychotic drugs Preclamol and OSU-6162, as well as for the preparation of two important intermediates in synthetic routes of the ovarian cancer monotherapeutic Niraparib