145 research outputs found

    Unlocking New Reactivities in Enzymes by Iminium Catalysis

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    The application of biocatalysis in conquering challenging synthesis requires the constant input of new enzymes. Developing novel biocatalysts by absorbing catalysis modes from synthetic chemistry has yielded fruitful new-to-nature enzymes. Organocatalysis was originally bio-inspired and has become the third pillar of asymmetric catalysis. Transferring organocatalytic reactions back to enzyme platforms is a promising approach for biocatalyst creation. Herein, we summarize recent developments in the design of novel biocatalysts that adopt iminium catalysis, a fundamental branch in organocatalysis. By repurposing existing enzymes or constructing artificial enzymes, various biocatalysts for iminium catalysis have been created and optimized via protein engineering to promote valuable abiological transformations. Recent advances in iminium biocatalysis illustrate the power of combining chemomimetic biocatalyst design and directed evolution to generate useful new-to-nature enzymes

    In Situ Acetaldehyde Synthesis for Carboligation Reactions

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    The enzyme 4-oxalocrotonate tautomerase (4-OT) can promis-cuously catalyze various carboligation reactions using acetalde-hyde as a nucleophile. However, the highly reactive nature ofacetaldehyde requires intricate handling, which can impede itsusage in practical synthesis. Therefore, we investigated threeenzymatic routes to synthesize acetaldehyde in situ in one-potcascade reactions with 4-OT. Two routes afforded practicalacetaldehyde concentrations, using an environmental pollu-tant,trans-3-chloroacrylic acid, or a bio-renewable, ethanol, asstarting substrate. These routes can be combined with 4-OTcatalyzed Michael-type additions and aldol condensations inone pot. This modular systems biocatalysis methodology pro-vides a stepping stone towards the development of larger arti-ficial metabolic networks for the practical synthesis of impor-tant chemical synthons

    Enantiocomplementary Michael Additions of Acetaldehyde to Aliphatic Nitroalkenes Catalyzed by Proline‐Based Carboligases

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    The blockbuster drug Pregabalin is widely prescribed for the treatment of painful diabetic neuropathy. Given the continuous epidemic growth of diabetes, the development of sustainable synthesis routes for Pregabalin and structurally related pharmaceutically active γ-aminobutyric acid (GABA) derivatives is of high interest. Enantioenriched γ-nitroaldehydes are versatile synthons for the production of GABA derivatives, which can be prepared through a Michael-type addition of acetaldehyde to α,β-unsaturated nitroalkenes. Here we report that tailored variants of the promiscuous enzyme 4-oxalocrotonate tautomerase (4-OT) can accept diverse aliphatic α,β-unsaturated nitroalkenes as substrates for acetaldehyde addition. Highly enantioenriched aliphatic ( R )- and ( S )-γ-nitroaldehydes were obtained in good yields using two enantiocomplementary 4-OT variants. Our results underscore the synthetic potential of 4-OT for the preparation of structurally diverse synthons for bioactive analogues of Pregabalin

    Selective Colorimetric “Turn-On” Probe for Efficient Engineering of Iminium Biocatalysis

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    The efficient engineering of iminium biocatalysis has drawn considerable attention, with many applications in pharmaceutical synthesis. Here, we report a tailor-made iminium-activated colorimetric "turn-on" probe, specifically designed as a prescreening tool to facilitate engineering of iminium biocatalysis. Upon complexation of the probe with the catalytic Pro-1 residue of the model enzyme 4-oxalocrotonate tautomerase (4-OT), a brightly colored merocyanine-dye-type structure is formed. 4-OT mutants that formed this brightly colored species upon incubation with the probe proved to have a substantial activity for the iminium-based Michael-type addition of nitromethane to cinnamaldehyde, whereas mutants that showed no staining by the probe exhibited no or very low-level "Michaelase" activity. This system was exploited in a solid-phase prescreening assay termed as activated iminium colony staining (AICS) to enrich libraries for active mutants. AICS prescreening reduced the screening effort up to 20-fold. After two rounds of directed evolution, two artificial Michaelases were identified with up to 39-fold improvement in the activity for the addition of nitromethane to cinnamaldehyde, yielding the target γ-nitroaldehyde product with excellent isolated yield (up to 95%) and enantiopurity (up to >99% ee). The colorimetric activation of the turn-on probe could be extended to the class I aldolase 2-deoxy-d-ribose 5-phosphate aldolase, implicating a broader application of AICS in engineering iminium biocatalysis

    Recent Applications of Carbon‐Nitrogen Lyases in Asymmetric Synthesis of Noncanonical Amino Acids and Heterocyclic Compounds

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    Carbon-nitrogen (C−N) lyases are enzymes that normally catalyze the cleavage of C−N bonds. Reversing this reaction towards carbon-nitrogen bond formation can be a powerful approach to prepare valuable compounds that could find applications in everyday life. This review focuses on recent (last five years) applications of native and engineered C−N lyases, either as stand-alone biocatalysts or as part of multienzymatic and chemoenzymatic cascades, in enantioselective synthesis of noncanonical amino acids and dinitrogen-fused heterocycles, which are useful tools for neurobiological research and important synthetic precursors to pharmaceuticals and food additives

    Enzymatic Oxy- and Amino-Functionalization in Biocatalytic Cascade Synthesis:Recent Advances and Future Perspectives

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    Biocatalytic cascades are a powerful tool for building complex molecules containing oxygen and nitrogen functionalities. Moreover, the combination of multiple enzymes in one pot offers the possibility to minimize downstream processing and waste production. In this review, we illustrate various recent efforts in the development of multi-step syntheses involving C-O and C-N bond-forming enzymes to produce high value-added compounds, such as pharmaceuticals and polymer precursors. Both in vitro and in vivo examples are discussed, revealing the respective advantages and drawbacks. The use of engineered enzymes to boost the cascades outcome is also addressed and current co-substrate and cofactor recycling strategies are presented, highlighting the importance of atom economy. Finally, tools to overcome current challenges for multi-enzymatic oxy- and amino-functionalization reactions are discussed, including flow systems with immobilized biocatalysts and cascades in confined nanomaterials.</p

    The broad amine scope of pantothenate synthetase enables the synthesis of pharmaceutically relevant amides

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    Pantothenate synthetase from Escherichia coli (PS(E. coli)) catalyzes the ATP-dependent condensation of (R)-pantoic acid and β-alanine to yield (R)-pantothenic acid (vitamin B(5)), the biosynthetic precursor to coenzyme A. Herein we show that besides the natural amine substrate β-alanine, the enzyme accepts a wide range of structurally diverse amines including 3-amino-2-fluoropropionic acid, 4-amino-2-hydroxybutyric acid, 4-amino-3-hydroxybutyric acid, and tryptamine for coupling to the native carboxylic acid substrate (R)-pantoic acid to give amide products with up to >99% conversion. The broad amine scope of PS(E. coli) enabled the efficient synthesis of pharmaceutically-relevant vitamin B(5) antimetabolites with excellent isolated yield (up to 89%). This biocatalytic amide synthesis strategy may prove to be useful in the quest for new antimicrobials that target coenzyme A biosynthesis and utilisation

    Enhancing the Peroxygenase Activity of a Cofactor‐Independent Peroxyzyme by Directed Evolution Enabling Gram‐Scale Epoxide Synthesis

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    Peroxygenases selectively incorporate oxygen into organic molecules making use of the environmentally friendly oxidant H(2)O(2) with water being the sole by‐product. These biocatalysts can provide ‘green’ routes for the synthesis of enantioenriched epoxides, which are fundamental intermediates in the production of pharmaceuticals. The peroxyzyme 4‐oxalocrotonate tautomerase (4‐OT), catalysing the epoxidation of a variety of α,β‐unsaturated aldehydes with H(2)O(2), is outstanding because of its independence from any cost‐intensive cofactor. However, its low‐level peroxygenase activity and the decrease in the enantiomeric excess of the corresponding α,β‐epoxy‐aldehydes under preparative‐scale conditions is limiting the potential of 4‐OT. Herein we report the directed evolution of a tandem‐fused 4‐OT variant, which showed an ∼150‐fold enhanced peroxygenase activity compared to 4‐OT wild type, enabling the synthesis of α,β‐epoxy‐aldehydes in milligram‐ and gram‐scale with high enantiopurity (up to 98 % ee) and excellent conversions. This engineered cofactor‐independent peroxyzyme can provide new opportunities for the eco‐friendly and practical synthesis of enantioenriched epoxides at large scale
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