Santos, Mariana; (2023) A study of chemoenzymatic and multi-enzymatic microreactor cascades for the synthesis of pharmaceutical compounds in continuous flow. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Biocatalytic cascades have emerged as efficient tools in the synthesis of valuable pharmaceutical compounds. Multi-enzymatic and chemoenzymatic cascades offer several advantages. Some examples are the ability to produce complex chiral molecules from simple building blocks, the reduced need for isolation of intermediates, and the potential to shift reaction equilibrium towards product synthesis. Their performance, however, requires the optimisation of several parameters to overcome incompatible reaction conditions and inhibitory interactions between the cascade components. Continuous flow microreactors, with their small reagent requirements, efficient mass and heat transfer and improved spatial control of reaction steps, are a promising technology for developing biocatalytic cascades. In this work, continuous flow techniques were explored in the development of two novel biocatalytic cascades. The first reaction investigated the coupling of a Diels Alder reaction, catalysed by aluminium chloride, with a transketolase-catalysed reaction for the synthesis of 1-(3,4-dimethyl-3-cyclohexen-1-yl)-1,3- dihydroxypropan-2-one. The different catalysts were combined thanks to the spatial separation of reaction steps afforded by microreactors. Optimisation of reaction conditions led to the complete conversion of substrates and a total process yield of 5 mM of product. Further process intensification strategies were developed to increase the cascade productivity. These included a numbering-up approach of identical microreactors and product purification methods via enzyme immobilisation and integration of an in-line tangential flow filtration. The second enzymatic cascade was designed to synthesise the only glycolytic metabolite commercially unavailable, 1,3-biphosphoglycerate. Several enzyme combinations and starting substrates were investigated under different operation modes. A three-enzyme cascade was established to realise the synthesis with in situ co-factor regeneration. The metabolite prepared in the cascade can serve as an effective substrate to phosphoglycerate kinase to produce adenosine triphosphate. Operational guidelines are proposed to assist in the efficient enzymatic preparation of 1,3-biphosphoglycerate for posterior isolation and use in scientific research.

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