Biocatalytic Arene Functionalization Using Integrated Unspecific Peroxygenase and Halohydrin Dehalogenase

In pharmaceuticals, aromatic structures are common motifs, underscoring the need for efficient and sustainable functionalization of arene compounds, particularly in late-stage functionalization. This study introduces a biocatalytic cascade employing unspecific peroxygenases (UPOs; PaDa-I mutant) and halohydrin dehalogenases (HHDHs), performed under batch conditions as a proof of concept. Significant advancements were achieved in the design of our microfluidic devices, where we incorporated HPLC screw fittings to minimize leakage and enhance compatibility with conventional flow equipment. To immobilize PaDa-I, we explored three covalent methods: (i) poly dopamine (PDA) surface coating, (ii) PDA with copolymerized polyethylenimine, and (iii) carbodiimide cross-linking. Among these, carbodiimide cross-linking achieved the highest activity yield (27%) under continuous-flow conditions, representing the most efficient immobilization reported to date for PaDa-I. To circumvent UPO instability toward hydrogen peroxide, we substituted it with ascorbic acid (AscA) for the epoxidation of styrene derivatives, improving PaDa-I stability to 6 days, achieving a yield of 37%, and maintaining productivity of 17 μM/h. Using EMIN340 and EMIN510 mutants from the commercial HHDH kit of Enzymaster facilitated the conversion of styrene oxide to N-phenyl-2-oxazolidinone, achieving a 1.3% yield over two steps. Notably, the use of AscA in the epoxidation showed a higher yield of 10.9% in the epoxide opening reaction toward N-phenyl-2-oxazolidinone compared to 4% using hydrogen peroxide. These findings underscore the potential of this biocatalytic cascade for further continuous-flow applications, offering improved enzyme stability and activity, with promising prospects for further optimization and broader implementation.