Stereopure amines are essential synthetic precursors. Selective catalysis is a key tool for stereoselective synthesis that is underpinned by both kinetic and thermodynamic effects. These effects become most interesting in reversible reactions when one product is kinetically favored and another thermodynamically favored, allowing the potential for a single catalyst to produce alternate products depending on reaction conditions. Transaminases are attractive catalysts for stereoselective synthesis of amines without transition metals. Substantial research has established the use of laboratory evolution to adapt enzymes, including transaminases, to synthesize stereopure products. In contrast, few studies have exploited kinetic and thermodynamic effects for stereodivergent, biocatalytic reactions. In this study, we report that the reversibility of PLP-dependent transaminases enables design of a single biocatalyst capable of synthesizing either diastereomer of a product with both high conversion and high diastereoselectivity. In particular, we investigated the whole cell, transamination of 2-,3-, and 4-substituted cyclohexanones. Our findings show that varying the concentrations of the amine donor and PLP cofactor changes the outcome of the reductive amination of 4-methylcyclohexanone from 95% conversion and &gt;10 : 1 cis : trans under pseudo-irreversible conditions to 70% conversion and &gt;10 : 1 trans : cis under reaction conditions that promote reversibility. Moreover, this diastereodivergent phenomenon extends to eight other 4-substituted substrates. 2- and 3-substituted cyclohexanones also react with high stereodivergence, leading to products that are both diastereo- and enantiopure. Supplementation of cells expressing transaminases with pyridoxine was further found to increase the effectiveness of the whole cell catalyst system. Finally, we found that mutagenesis of the transaminase can be combined with kinetic and thermodynamic principles to both further increase the kinetic preference of a catalyst toward the kinetic product and to increase the rate of equilibration toward the thermodynamic product. Reversibility is a common property of reductive, enzymatic reactions that form stereocenters, reactions for which analogous chemical methods are often irreversible. Beyond the synthesis of amines disclosed here, these findings suggest the potential to exploit kinetic and thermodynamic effects to improve the selectivity of other biocatalytic processes.

Beyond mutagenesis: Highly stereodivergent biocatalytic synthesis of amines via kinetic and thermodynamic control

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