Abstract
Cooperative photobiocatalysis is a useful strategy for achieving stereoselective intermolecular radical reactions that are not known in either biology or chemistry. The diastereoselective and enantioselective synthesis of cyclic non-canonical amino acids remains challenging using established methods. Here we report a multienzyme photobiocatalytic cascade to stereoselectively prepare polysubstituted unnatural prolines. We engineered an underexploited class of pyridoxal 5'-phosphate aldolases as new-to-nature radical carboligases to catalyse the decarboxylative C-C coupling of aspartic acid, furnishing imine-containing azacyclic non-canonical amino acids. High-throughput screening of metagenomic imine reductases led to the development of diastereoselective biocatalytic reduction and dynamic kinetic asymmetric transformation of cyclic imines, providing optically pure unnatural prolines featuring an elusive 2,5-anti stereochemistry with up to three stereocentres. Beyond its synthetic utility, this study established a new mode of radical pyridoxal enzymology by leveraging open-shell enamine catalysis, opening up avenues for developing new free radical reactions.