A key aim of biocatalysis is to mimic the ability of eukaryotic cells to carry out multistep cascades in a controlled and selective way. As biocatalytic cascades get more complex, reactions become unattainable under typical batch conditions. Here a number of continuous flow systems were used to overcome batch incompatibility, thus allowing for successful biocatalytic cascades. As proof‐of‐principle, reactive carbonyl intermediates were generated in situ using alcohol oxidases, then passed directly to a series of packed‐bed modules containing different aminating biocatalysts which accordingly produced a range of structurally distinct amines. The method was expanded to employ a batch incompatible sequential amination cascade via an oxidase/transaminase/imine reductase sequence, introducing different amine reagents at each step without cross‐reactivity. The combined approaches allowed for the biocatalytic synthesis of the natural product 4O‐methylnorbelladine.

Synthesis of Cbz-protected 3-aminopiperidine and 3-aminoazepane using a multi-enzyme cascade consisting of galactose oxidase and imine reductase variants.

Iminosugar scaffolds are highly sought-after pharmaceutical targets, but their chemical synthesis is lengthy and can suffer from poor scalability and purification. Here we report protecting-group-free chemoenzymatic and biocatalytic cascades to synthesize iminosugars from sugar-derived aminopolyols in two steps. Using galactose oxidase variant F 2 followed by a chemical or enzymatic reduction provided an efficient one-pot route to these targets, with product formation >70%. Key to success of this strategy was the application of genome mining, which identified bacterial shikimate dehydrogenases as promiscuous iminosugar reductases. The cell-free protocols allowed for isolation of highly polar iminosugar products from biotransformations in a single step through development of a gradient-elution cation exchange purification. The two-step pathway provides a short synthetic route that can be used as a cell-free platform for broader iminosugar synthesis.

Amino-polyols represent attractive chemical building blocks but can be challenging to synthesize because of the high density of asymmetric functionalities and the need for extensive protecting-group strategies. Here we present a three-component strategy for the stereoselective enzymatic synthesis of amino-diols and amino-polyols using a diverse set of prochiral aldehydes, hydroxy ketones, and amines as starting materials. We were able to combine biocatalytic aldol reactions, using variants of D-fructose-6-phosphate aldolase (FSA), with reductive aminations catalyzed by IRED-259, identified from a metagenomic library. A two-step process, without the need for intermediate isolation, was developed to avoid cross-reactivity of the carbonyl components. Stereoselective formation of the 2R,3R,4R enantiomers of amino-polyols was observed and confirmed by X-ray crystallography.

A key aim of biocatalysis is to mimic the ability of eukaryotic cells to carry out multistep cascades in a controlled and selective way. As biocatalytic cascades get more complex, reactions become unattainable under typical batch conditions. Here a number of continuous flow systems were used to overcome batch incompatibility, thus allowing for successful biocatalytic cascades. As proof‐of‐principle, reactive carbonyl intermediates were generated in situ using alcohol oxidases, then passed directly to a series of packed‐bed modules containing different aminating biocatalysts which accordingly produced a range of structurally distinct amines. The method was expanded to employ a batch incompatible sequential amination cascade via an oxidase/transaminase/imine reductase sequence, introducing different amine reagents at each step without cross‐reactivity. The combined approaches allowed for the biocatalytic synthesis of the natural product 4O‐methylnorbelladine.