Designing biocatalysts for non-natural carboligations

Building complex compounds such as fine chemicals and pharmaceuticals by linking carbon-carbon bonds is at the core of organic synthesis. However, current methods to construct carbon-carbon bonds often have significant shortcomings, such as the use of hazardous substances or the generation of toxic waste. Biocatalysis, the use of enzymes in organic synthesis, potentially offers a more eco-friendly and sustainable approach for carbon-carbon bond formation. However, many powerful transformations known in chemistry are not catalyzed by natural enzymes.The work described in the PhD thesis of Andreas Kunzendorf focuses on the discovery and engineering of new enzymes for several highly interesting carbon-carbon bond-forming reactions. The research yielded new biocatalysts for the synthesis of important cyclopropanes and efficient enzymes for the production of building blocks for pharmaceutically active γ-aminobutyric acids via extensive enzyme optimisation using directed evolution. The optimized enzyme variants could not only perform the reactions much faster but also provided the products with high selectivity. The results of this work are a stepping-stone towards more sustainable synthesis of pharmaceuticals and provide exciting opportunities to develop new enzymatic reactions not known in nature

In Situ Acetaldehyde Synthesis for Carboligation Reactions

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

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