Biocatalytic Cascades toward Iminosugar Scaffolds Reveal Promiscuous Activity of Shikimate Dehydrogenases

Abstract

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.

The shikimate pathway: gateway to metabolic diversity

“…78 To date, the processing of shikimate into reduced and dehydrated cyclohexanes and cyclohexenes are the known routes to repurpose shikimate into natural product scaffolds, however recent investigations have demonstrated the flexibility of shikimate dehydrogenases in the biocatalytic synthesis of iminosugars. 79…”

“…78 To date, the processing of shikimate into reduced and dehydrated cyclohexanes and cyclohexenes are the known routes to repurpose shikimate into natural product scaffolds, however recent investigations have demonstrated the exibility of shikimate dehydrogenases in the biocatalytic synthesis of iminosugars. 79 Shikimate itself is rarely directly incorporated into natural product scaffolds, with few notable exceptions such as the chlorogenic acids found in plants, as discussed in Section 3 (Fig. 5).…”

Redox Out of the Box: Catalytic Versatility Across NAD(P)H‐Dependent Oxidoreductases

“…[30] Recently, AroE was postulated to reduce C=N bonds in iminosugar imines and was successfully combined with galactose oxidase to access imino sugar scaffolds from aminopolyols (Scheme 3B). [31] However, given the possibility for an Amadori rearrangement of the α-hydroxy-imine to an α-amino-ketone, it cannot be ruled out that the enzyme actually reduced a C=O bond. [31]…”

“…[31] However, given the possibility for an Amadori rearrangement of the α-hydroxy-imine to an α-amino-ketone, it cannot be ruled out that the enzyme actually reduced a C=O bond. [31]…”

Enzyme‐Triggered Reactions for the Synthesis of Organic Molecules

“…In a one-pot reaction, Swanson et al showed that the galactose oxidase F 2 variant could catalyse the primary oxidation of unprotected aminopolyols 25, followed by a chemical reduction step for the preparation of various iminosugars 26 (Scheme 4d). [13] The intermediate 27 leading to the iminosugars with a deoxy-ribo or arabino configuration could be reduced by Shikimate dehydrogenases, possibly via an Amadori rearrangement, and subsequent enzyme-catalysed asymmetric ketone reduction of the α-amino carbonyl moiety. The GOase variants F 2 and M 3-5 have furthermore been used for the enzyme-triggered formation of non-sugar compounds, such as lactams 28, 29, and 30 (Scheme 4e).…”