Scoping the Enantioselective Desymmetrization of a Poorly Water-Soluble Diester by Recombinant Pig Liver Esterase

Abstract

Previously, the biocatalytic desymmetrization of dimethyl cyclohex-4-ene-cis-1,2-dicarboxylate to (1S,2R)-1-(methoxycarbonyl)cyclohex-4-ene-2-carboxylic acid, an important intermediate toward the synthesis of biologically active molecules, had been well-characterized in terms of pH and temperature optima and several aspects of process performance. Eventually this promising reaction could convert 200 mM (40 g·L–1) of substrate with >99.5% ee using the recombinant pig liver esterase, ECS-PLE06, at a scale of 8.8 L. However, the precise influence of substrate concentration and the poorly water-soluble nature of the substrate (approximately 60 mM in water at 25 °C for structurally similar dimethyl 1,4-cyclohexanedicarboxylate) remained elusive. Therefore, this work focuses on using a recently published methodology based on reaction trajectory analysis to identify mass transfer limitations in this reaction. With the constraints of mass transfer on space-time yield considered, it was possible to evaluate and improve biocatalyst yield (mass of product per mass of biocatalyst) through the use of higher substrate concentrations. Ultimately the complete conversion of approximately 75 g·L–1 substrate was achieved in 3.65 h yielding an excellent productivity of 20 g·L–1·h–1 with a biocatalyst yield of 4.36 g·gbiocat–1. This work also highlights the simplicity of applying a reaction trajectory analysis methodology and the importance of scale during reaction characterizations, and it identifies future directions for reaction improvement to address substrate supply and product inhibition/deactivation.