Covalently Immobilized 2‐Deoxyribose‐5‐phosphate Aldolase (DERA) for Biocatalysis in Flow: Utilization of the 3‐Hydroxyaldehyde Intermediate in Reaction Cascades

Abstract

C−C bond forming enzymes, such as 2‐deoxyribose‐5‐phosphate aldolase (DERA), are an important class of enzymes in synthesis. The use of DERA, with its reaction routes to the double aldol product, is an established method for producing statin side chains in industry. Herein, the selective synthesis of 3‐hydroxyaldehydes as single aldol products of DERA is presented. Successful covalent immobilization of a suitable Escherichia coli (Ec) DERA variants with C47M and C47V exchanges, respectively, enabled their use in continuous synthesis. Precise reaction control using flow chemistry made the selective formation of 3‐hydroxyoctanal from hexanal possible. For optimization, a systematic approach of design of experiment (DoE) was used, resulting in 90 % conversion. Furthermore, five two‐step reaction systems have been established to scavenge the reactive single aldol product combining biocatalysis in flow mode and a consecutive chemical reactions step either in flow (continuous flow mode) or in batch (semi‐continuous flow mode).

Application of Immobilized Enzymes in Flow Biocatalysis for Efficient Synthesis

“…Pietruszka's group published an effective protocol for using the immobilized variant of DERA in the flow cascade process to synthesize 3-hydroxyaldehyde and then produce the desired α,β-unsaturated octenal, a known flavor component found in fruits and fungi (Figure 26). 112 The first step is the reaction to produce (R)-3-hydroxyoctanal by the covalently immobilized DERA variant from E. coli DERA EC -C47 M via the Halotag system. The second step is the elimination of water via the packed-bed reactor containing an alkaline resin (Amberlyst A-21).…”

“…Moreover, the immobilized enzymes provide convenience of operation for this process. In addition, flow cascade reactions are available not only for multiple incompatible biocatalyst-catalyzed reactions, such as the ALDH Tt -LDH reactor described by Magner and co-workers but also for the combination with chemical catalysts or other technologies, such as metal catalysts, photocatalysis, etc. ,− …”

Trendbericht Organische Chemie 2023

“…Pietruszka immobilisierte die 2-Deoxyribose-5-phosphataldolase (DERA) mit einem Halotag und erreichte damit die doppelte Aldoladdition, wobei chirale 3,5-Dihydroxyaldehyde entstehen. 61) Ein Vorteil der Flowsynthese ist, dass sich hochreaktive Intermediate oder instabile Produkte sicher herstellen und weiterverarbeiten lassen. Nagaki synthetisierte durch Elektrolyse in einem Flow-Mikroreaktor Carbokationen, die innerhalb von Millisekunden in folgenden Reaktionen mit Nukleophilen abgefangen werden.…”