High‐Throughput Discovery of Stereodivergent Sulfoxide Synthases

ABSTRACT Chiral sulfoxides are important motifs in pharmaceuticals, yet their asymmetric synthesis remains challenging due to the need for stereocontrol. While bio‐oxidation provides an attractive route, the lack of robust high‐throughput screening methods hinders the development of enantioselective sulfoxide synthases. Herein, we developed a high‐throughput assay combining sulfoxide reductases (MsrA/B) with a DL‐dithiothreitol and 5,5’‐dithiobis (2‐nitrobenzoic acid) chromogenic system. This platform enables the simultaneous assessment of both catalytic efficiency and enantioselectivity, with colorimetric signals showing a linear correlation ( R 2 = 0.99) against HPLC‐validated enantiomeric excess. As a proof of concept, we screened 2880 engineered variants of recombinant unspecific peroxygenase from Agrocybe aegerita (r Aae UPO), a known thioether‐oxidizing enzyme. This method allows the identification of mutants with complementary enantioselectivity (95.2% ee ( R ) or 10.3% ee ( S )). In addition, the modular design of this assay—exploiting the stereo‐complementarity of MsrA/B toward sulfoxides—provides a general framework for developing other stereoselective oxidoreductases for thioether oxidation.