Continuous Flow Biocatalytic Reductive Amination by Co-Entrapping Dehydrogenases with Agarose Gel in a 3D-Printed Mould Reactor.

Herein, we show how the merge of biocatalysis with flow chemistry aided by 3D-printing technologies can facilitate organic synthesis. This concept was exemplified for the reductive amination of benzaldehyde catalysed by co-immobilised amine dehydrogenase and formate dehydrogenase in a continuous flow micro-reactor. For this purpose, we investigated enzyme co-immobilisation by covalent binding, or ion-affinity binding, or entrapment. Entrapment in an agarose hydrogel turned out to be the most promising solution for this biocatalytic reaction. Therefore, we developed a scalable and customisable approach whereby an agarose hydrogel containing the co-entrapped dehydrogenases was cast in a 3D-printed mould. The reactor was applied to the reductive amination of benzaldehyde in continuous flow over 120 h and afforded 47 % analytical yield and a space-time yield of 7.4 g L day-1 using 0.03 mol% biocatalysts loading. This work also exemplifies how rapid prototyping of enzymatic reactions in flow can be achieved through 3D-printing technology.

Enantioselective Biocascade Catalysis with a Single Multifunctional Enzyme.

Asymmetric catalytic cascade processes offer direct access to complex chiral molecules from simple substrates and in a single step. In biocatalysis, cascades are generally designed by combining multiple enzymes, each catalyzing individual steps of a sequence. Herein, we report a different strategy for biocascades based on a single multifunctional enzyme that can promote multiple stereoselective steps of a domino process by mastering distinct catalytic mechanisms of substrate activation in a sequential way. Specifically, we have used an engineered 4-oxalocrotonate tautomerase (4-OT) enzyme with the ability to form both enamines and iminium ions and combine their mechanisms of catalysis in a complex sequence. This approach allowed us to activate aldehydes and enals toward the synthesis of enantiopure cyclohexene carbaldehydes. The multifunctional 4-OT enzymes could promote both a two-component reaction and a triple cascade characterized by different mechanisms and activation sequences.

Kinetic Resolution of Racemic Primary Amines Using Geobacillus stearothermophilus Amine Dehydrogenase Variant.

A NADH-dependent engineered amine dehydrogenase from Geobacillus stearothermophilus (LE-AmDH-v1) was applied together with a NADH-oxidase from Streptococcus mutans (NOx) for the kinetic resolution of pharmaceutically relevant racemic α-chiral primary amines. The reaction conditions (e. g., pH, temperature, type of buffer) were optimised to yield S-configured amines with up to >99 % ee.

Synthetic Evidence of the Amadori-Type Alkylation of Biogenic Amines by the Neurotoxic Metabolite Dopegal.

The neurotransmitter metabolite 3,4-dihydroxy-phenylglycolaldehyde (dopegal) damages neurons and the myocardium by protein cross-linking, resulting in conglomerations and cell death. We investigated this process on a synthetic scale, leading to the discovery of an Amadori-type rearrangement of dopegal in the reaction with several amino acids and neuropeptides. This alkylation also occurs with neurotransmitters, suggesting an influence of dopegal on neurochemical processes. The rearrangement occurs readily under physiological conditions.

Catalytic Promiscuity of Galactose Oxidase: A Mild Synthesis of Nitriles from Alcohols, Air, and Ammonia.

We report an unprecedented catalytically promiscuous activity of the copper-dependent enzyme galactose oxidase. The enzyme catalyses the one-pot conversion of alcohols into the related nitriles under mild reaction conditions in ammonium buffer, consuming ammonia as the source of nitrogen and dioxygen (from air at atmospheric pressure) as the only oxidant. Thus, this green method does not require either cyanide salts, toxic metals, or undesired oxidants in stoichiometric amounts. The substrate scope of the reaction includes benzyl and cinnamyl alcohols as well as 4- and 3-pyridylmethanol, giving access to valuable chemical compounds. The oxidation proceeds through oxidation from alcohol to aldehyde, in situ imine formation, and final direct oxidation to nitrile.

The ALDH21 gene found in lower plants and some vascular plants codes for a NADP+ -dependent succinic semialdehyde dehydrogenase.

Lower plant species including some green algae, non-vascular plants (bryophytes) as well as the oldest vascular plants (lycopods) and ferns (monilophytes) possess a unique aldehyde dehydrogenase (ALDH) gene named ALDH21, which is upregulated during dehydration. However, the gene is absent in flowering plants. Here, we show that ALDH21 from the moss Physcomitrella patens codes for a tetrameric NADP+ -dependent succinic semialdehyde dehydrogenase (SSALDH), which converts succinic semialdehyde, an intermediate of the γ-aminobutyric acid (GABA) shunt pathway, into succinate in the cytosol. NAD+ is a very poor coenzyme for ALDH21 unlike for mitochondrial SSALDHs (ALDH5), which are the closest related ALDH members. Structural comparison between the apoform and the coenzyme complex reveal that NADP+ binding induces a conformational change of the loop carrying Arg-228, which seals the NADP+ in the coenzyme cavity via its 2'-phosphate and α-phosphate groups. The crystal structure with the bound product succinate shows that its carboxylate group establishes salt bridges with both Arg-121 and Arg-457, and a hydrogen bond with Tyr-296. While both arginine residues are pre-formed for substrate/product binding, Tyr-296 moves by more than 1 Å. Both R121A and R457A variants are almost inactive, demonstrating a key role of each arginine in catalysis. Our study implies that bryophytes but presumably also some green algae, lycopods and ferns, which carry both ALDH21 and ALDH5 genes, can oxidize SSAL to succinate in both cytosol and mitochondria, indicating a more diverse GABA shunt pathway compared with higher plants carrying only the mitochondrial ALDH5.