3D‐Printed Phenacrylate Decarboxylase Flow Reactors for the Chemoenzymatic Synthesis of 4‐Hydroxystilbene

Abstract

Continuous flow systems for chemical synthesis are becoming a major focus in organic chemistry and there is a growing interest in the integration of biocatalysts due to their high regio‐ and stereoselectivity. Methods established for 3D bioprinting enable the fast and simple production of agarose‐based modules for biocatalytic reactors if thermally stable enzymes are available. We report here on the characterization of four different cofactor‐free phenacrylate decarboxylase enzymes suitable for the production of 4‐vinylphenol and test their applicability for the encapsulation and direct 3D printing of disk‐shaped agarose‐based modules that can be used for compartmentalized flow microreactors. Using the most active and stable phenacrylate decarboxylase from Enterobacter spec. in a setup with four parallel reactors and a subsequent palladium(II) acetate‐catalysed Heck reaction, 4‐hydroxystilbene was synthesized from p‐coumaric acid with a total yield of 14.7 % on a milligram scale. We believe that, due to the convenient direct immobilization of any thermostable enzyme and straightforward tuning of the reaction sequence by stacking of modules with different catalytic activities, this simple process will facilitate the establishment and use of cascade reactions and will therefore be of great advantage for many research approaches.

Flow Chemistry in Contemporary Chemical Sciences: A Real Variety of Its Applications

“…It offers the production of extremely complex geometries; nevertheless, only a limited number of polymeric materials can be used with this technique, and some additional treatment, e.g., electron beam irradiation can be helpful in obtaining satisfactory mechanical properties of 3D-printed elements [283]. The examples of 3D-printed modules for flow synthesis include different flow-through reactors [51,284,285] and microfluidic chips for the synthesis of Ag and Au nanoparticles [286]. A 3D-printed microfluidic system was also produced for the generation of microdroplets, which can be used when creating functionalized microparticles [287].…”

Enzyme Scaffolds with Hierarchically Defined Properties via 3D Jet Writing

“…[3] In addition, enzyme immobilization enables the use in continuous flow reactors, which can lead to increased throughput. [4,5] The physical and chemical properties of the support material as well as the nature of the enzyme are decisive factors in enzyme immobilization. [6] Enzyme immobilization methods can be classified into three different categories: i) intermolecular crosslinking between the enzyme and a filler molecule, ii) conjugation of the enzyme to a support, and iii) physical entrapment of the enzyme inside of a matrix.…”

A Phenolic Acid Decarboxylase-Based All-Enzyme Hydrogel for Flow Reactor Technology

“…The sequences of the primers can be found in Table 1. pET_PAD-His was used as a template for further cloning steps [30].…”

“…The sequences of the primers can be found in Table 1. pET_PAD-His was used as a template for further cloning steps [30]. pET_PAD-SC-His: The backbone encoding for a N-terminal PAD and C-terminal 6× His-Tag separated by a glycine spacer was amplified using primers EM01 and EM02 with pET_PAD-His as the template.…”

“…The versatility of PADs has been demonstrated by integration of the decarboxylation reaction into biocatalytic [6,27] and chemical [28][29][30] reaction cascades. However, limitations can arise due to the product inhibition [28,29] frequently observed for this class of enzymes.…”