Dual-Function Cosubstrates Enable Threonine Dehydrogenase-Driven Chemoenzymatic Pyrrole Synthesis

Preparative biocatalytic synthesis of substituted pyrroles remains challenging despite recent advances in enzymatic α-aminoketone generation. In this study, we report a concurrent chemoenzymatic cascade to disubstituted pyrroles based on threonine dehydrogenase (ThrDH)-catalyzed generation of aminoacetone from l-threonine that is intercepted in situ by Knorr pyrrole condensation with β-dicarbonyl compounds. To overcome cofactor limitations at high substrate concentrations, Escherichia coli ThrDH was coupled to an alcohol dehydrogenase from Rhodococcus ruber DSM 44541 (ADH-A), enabling dual-function of the β-dicarbonyl cosubstrate as both a hydride acceptor and pyrrole building-block. The whole-cell catalyst tolerated up to 8% (v/v) β-dicarbonyl and afforded pyrrole products in up to 90% yield and space-time yields of 4.5 g L^-1 h^-1. Preparative synthesis on a 100 mL scale furnished 4.3 g of pyrrole (279 mM, 93% isolated yield). Among the products obtained, the cascade provides direct access to a reported sunitinib intermediate, illustrating its potential utility for pharmaceutical synthesis. This work establishes ThrDHs as outstanding biocatalysts for heterocycle synthesis and demonstrates that amino acid feedstocks can be efficiently converted into substituted pyrroles through a scalable chemoenzymatic cascade.