A methodology for cascade selection for co-product removal in the ω-transaminase system

Production of chiral amines using transaminases has indeed been proposed recently as an interesting alternative to conventional methods to help in the synthesis of many new pharmaceuticals. Two reaction strategies have been demonstrated: kinetic resolution and asymmetric synthesis. The latter approach has the advantage that the theoretical yield is 100% compared to 50% for the former [1]. However, a major challenge for asymmetric synthesis is the unfavourable thermodynamic equilibrium for many of the most interesting reactions. Meeting the feasibility criteria that are typical for most pharmaceutical processes can only be achieved by selectively removing the product and/or co-product formed during the reaction (so called in-situ (co)product removal (IS(C)PR)). Several different alternative co-product removal strategies have been suggested, all of which have different impacts on the overall process. Among others, one of the most promising strategies is to use a second enzyme reaction to remove the co-product in an enzymatic cascade [2]. Currently there are no decision tools available to help select appropriate cascade systems for process implementation.
In the current work a methodology for choosing a feasible cascade system that will remove co-product to meet process requirements under process relevant conditions will be presented. Decisions are based on thermodynamic constraints, kinetics, selectivity, stability, pH change, cascade enzyme compatibility and downstream processing. The methodology has been applied to an ω-transaminase system which is thermodynamically challenged and enzymatic ISCPR is deployed to shift the equilibrium. The enzymes proposed for co-product removal are dehydrogenases: lactate dehydrogenase (EC 1.1.1.27), alanine dehydrogenase (EC 1.4.1.1), yeast alcohol dehydrogenase (EC 1.1.1.1); pyruvate decarboxylase (EC 4.1.1.1), acetolactate synthase (EC 2.2.1.6) and as co-factor recycling enzymes: glucose dehydrogenase (EC 1.1.1.47), formate dehydrogenase (EC 1.2.1.2) and phosphite dehydrogenase (EC 1.20.1.1). The methodology gives an insight into the constraints of different cascade systems and is the basis for process set-up of selected cascades. Experimental and calculated data will be used to illustrate the methodology.