Process Evaluation Tools for Enzymatic Cascades Welcome Message

Biocatalysis is attracting significant attention from both academic and industrial scientists due to the excellent capability of enzyme to catalyse selective reactions. Recently, much interest has been shown in the application of enzymatic cascades as a useful tool in organic synthesis to synthesize valuable compounds (e.g. chiral molecules) especially as pharmaceutical intermediates and to assist complex reactions that otherwise has problems as single step system.

Despite this interest, process evaluation of many enzymatic cascades has only rarely been reported in the search for process improvement and implementation. Hence, the goal of this thesis is to evaluate the process concepts in enzymatic cascades in a systematic manner, using tools such as thermodynamic and kinetic analysis.

Three relevant case studies have been used to exemplify the approach. In the first case study, thermodynamic and kinetic studies were used to evaluate the favourability of a redox neutral cascade for the asymmetric amination of alcohols to their corresponding chiral amines. This synthetic cascade is potentially attractive since it synthesizes valuable chiral molecule from cheap raw materials as well as maximising the atom economy.

The scheme consists of two primary enzymes (alcohol dehydrogenase and ω-transaminase) that are directly involved in the main synthesis. Alanine dehydrogenase was introduced as a secondary enzyme to regenerate the co-factor NAD+ and co-substrate alanine in situ as well as to shift the equilibrium positions in the main syntheses.

In principle, this strategy could successfully achieve high conversion, using ammonia as the sole reagent used in excess to drive the conversion. The findings herein indicate that quantitatively the possibilities for improving the conversion of thermodynamically limited reactions are not only via application of enzyme coupling reactions (coupling the unfavourable reaction with an energetically favourable reaction) but also by matching the relative reaction rates between the interconnecting enzymes.

When the reaction steps are independent in a cascade, the kinetics can be controlled in a highly efficient way to achieve a sufficiently favourable conversion to a given target product. This is exemplified in the second case study, in the kinetic modelling of the formation of 2-ketoglutarate from glucoronate, the second case study.

This cascade consists of 4 enzymes (uronate dehydrogenase, glucarate dehydratase, keto-deoxy-d-galactarate dehydratase and α-ketoglutaric semialdehyde dehydrogenase) run in that order to successfully achieve high conversion. Finally, a third case study was used to explore the effect of activity-coefficients in enzyme-catalysed reactions.

Frequently, the ‘apparent’ or (concentration-based) equilibrium constant (Κ′), instead of activity-based equilibrium constant, was used to describe reaction equilibria of biological systems. It is assumed that the reactant activity is equal to the respective reactant molar concentration at equilibrium since many reactions operate in dilute aqueous solutions and thus neglect the activity coefficient effect.

The effect of such assumption was therefore tested with the cyclohexanone amination with (S)-1-phenylethylamine catalysed by ω-transaminase. The findings showed that the activity coefficients of the components significantly deviate from unity, indicating its non-ideal behaviour in the reaction medium.

Hence, thermodynamic and kinetic analyses are powerful tools to evaluate and to achieve workable cascades for non-natural pathways. Additionally, more meaningful equilibrium data from enzyme-catalysed reactions can be a useful way to determine the effectiveness of a given cascade strategy.