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

Biocatalytic Asymmetric Cyclopropanations via Enzyme‐bound Iminium Ion Intermediates

Cyclopropane rings are an important structural motif frequently found in many natural products and pharmaceuticals. Commonly, biocatalytic methodologies for the asymmetric synthesis of cyclopropanes rely on repurposed or artificial heme enzymes. Here, we engineered an unusual cofactor‐independent cyclopropanation enzyme based on a promiscuous tautomerase for the enantioselective synthesis of various cyclopropanes via the nucleophilic addition of diethyl 2‐chloromalonate to α,β‐unsaturated aldehydes. The engineered enzyme promotes formation of the two new carbon‐carbon bonds with excellent stereocontrol over both stereocenters, affording the desired cyclopropanes with high diastereo‐ and enantiopurity (d.r. up to 25:1; e.r. up to 99:1). Our results highlight the usefulness of promiscuous enzymes for expanding the biocatalytic repertoire for non‐natural reactions

Gene Fusion and Directed Evolution to Break Structural Symmetry and Boost Catalysis by an Oligomeric C‐C Bond‐Forming Enzyme

Gene duplication and fusion are among the primary natural processes that generate new proteins from simpler ancestors. Here we adopted this strategy to evolve a promiscuous homohexameric 4-oxalocrotonate tautomerase (4-OT) into an efficient biocatalyst for enantioselective Michael reactions. We first designed a tandem-fused 4-OT to allow independent sequence diversification of adjacent subunits by directed evolution. This fused 4-OT was then subjected to eleven rounds of directed evolution to give variant 4-OT(F11), which showed an up to 320-fold enhanced activity for the Michael addition of nitromethane to cinnamaldehydes. Crystallographic analysis revealed that 4-OT(F11) has an unusual asymmetric trimeric architecture in which one of the monomers is flipped 180° relative to the others. This gene duplication and fusion strategy to break structural symmetry is likely to become an indispensable asset of the enzyme engineering toolbox, finding wide use in engineering oligomeric proteins

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

Using mutability landscapes of a promiscuous tautomerase to guide the engineering of enantioselective Michaelases

The Michael-type addition reaction is widely used in organic synthesis for carbon-carbon bond formation. However, biocatalytic methodologies for this type of reaction are scarce, which is related to the fact that enzymes naturally catalysing carbon-carbon bond-forming Michael-type additions are rare. A promising template to develop new biocatalysts for carbon-carbon bond formation is the enzyme 4-oxalocrotonate tautomerase, which exhibits promiscuous Michael-type addition activity. Here we present mutability landscapes for the expression, tautomerase and Michael-type addition activities, and enantioselectivity of 4-oxalocrotonate tautomerase. These maps of neutral, beneficial and detrimental amino acids for each residue position and enzyme property provide detailed insight into sequence-function relationships. This offers exciting opportunities for enzyme engineering, which is illustrated by the redesign of 4-oxalocrotonate tautomerase into two enantiocomplementary 'Michaelases'. These 'Michaelases' catalyse the asymmetric addition of acetaldehyde to various nitroolefins, providing access to both enantiomers of γ-nitroaldehydes, which are important precursors for pharmaceutically active γ-aminobutyric acid derivatives

Dichotomy between RIP1- and RIP3-mediated necroptosis in tumor necrosis factor-α-induced shock

Tumor necrosis factor receptor (TNFR) signaling may result in survival, apoptosis or programmed necrosis. The latter is called necroptosis if the receptor-interacting protein 1 (RIP1) inhibitor necrostatin-1 (Nec-1) or genetic knockout of RIP3 prevents it. In the lethal mouse model of TNFα-mediated shock, addition of the pan-caspase inhibitor zVAD-fmk (zVAD) accelerates time to death. Here, we demonstrate that RIP3-deficient mice are protected markedly from TNFα-mediated shock in the presence and absence of caspase inhibition. We further show that the fusion protein TAT-crmA, previously demonstrated to inhibit apoptosis, also prevents necroptosis in L929, HT29 and FADD-deficient Jurkat cells. In contrast to RIP3-deficient mice, blocking necroptosis by Nec-1 or TAT-crmA did not protect from TNFα/zVAD-mediated shock, but further accelerated time to death. Even in the absence of caspase inhibition, Nec-1 application led to similar kinetics. Depletion of macrophages, natural killer (NK) cells, granulocytes or genetic deficiency for T lymphocytes did not influence this model. Because RIP3-deficient mice are known to be protected from cerulein-induced pancreatitis (CIP), we applied Nec-1 and TAT-crmA in this model and demonstrated the deterioration of pancreatic damage upon addition of these substances. These data highlight the importance of separating genetic RIP3 deficiency from RIP1 inhibition by Nec-1 application in vivo and challenge the current definition of necroptosis