Biotechnological production, isolation and characterisation of (2R,3S)‐2,3‐dihydroxy‐2,3‐dihydrobenzoate

Abstract

Bacterial Rieske non‐heme iron oxygenases catalyse the transformation of a wide range of aromatic compounds to vicinal cis ‐dihydrodiols. Such compounds have been successfully applied in chemoenzymatic synthetic routes for, for example, pharmaceuticals, natural products and polymers. In the case of benzoate, only (1 S ,2 R )‐ cis ‐1,2‐dihydroxy‐2‐hydrobenzoate is readily accessible via enzymatic transformation, but not the regioisomeric cis ‐2,3‐dihydroxy‐2,3‐dihydrobenzoate (2,3‐DD) or cis ‐3,4‐dihydroxy‐3,4‐dihydrobenzoate. While trace amounts of putative cis ‐2,3‐DD have been obtained before by using p ‐cumate 2,3‐dioxygenase (PCDO) or a combination of chlorobenzene dioxygenase and nitrilase, none of these approaches enabled its production and isolation at a greater scale for potential use as a chiral building block in organic synthesis. We here provide a protocol for biotransformation of benzoate yielding (2 R ,3 S )‐2,3‐dihydroxy‐2,3‐dihydrobenzoate using the PCDO of Pseudomonas citronellolis strain EB200 with negligible formation of side products. An isolation procedure suitable for production of the 2,3‐DD sodium salt monohydrate at high purity (> 95%) at a gram scale, and a comprehensive characterisation of this novel metabolite is given.