Mix‐to‐Prepare Dicarboxylic Acid‐Based Decoy Molecules Enable Wild‐Type P450BM3‐Catalyzed Oxidation of Non‐Native Substrates

ABSTRACT Decoy molecules, which are substrate analogues, serve as enabling additives that unlock the catalytic potential of cytochrome P450BM3 toward non‐native substrates; however, their broader application has been limited by the requirement for prior chemical synthesis. In this study, we report amic acids as a new scaffold for decoy molecules, inspired by their structural similarity to the native P450BM3 substrate, N ‐palmitoylglycine. Amic acid‐based decoy molecules were rapidly prepared by simple mixing of cyclic anhydrides and amines and were directly evaluated without purification, enabling rapid screening. Using this approach, phthalic acid‐based decoy molecules were identified that promoted not only benzene hydroxylation but also toluene hydroxylation and styrene epoxidation, while also expanding the substrate scope to a broader range of aromatic and aliphatic compounds. X‐ray crystallographic analysis further revealed that the obtained decoy molecules closely mimic the native substrate within the active site of P450BM3. Notably, in whole‐cell benzene hydroxylation, phenol production nearly 3 mM was achieved, and comparable activity was observed even when unpurified decoy molecule solutions prepared within tens of minutes were used. Owing to their facile preparation and ease of use, amic acid‐based decoy molecules offer a generally applicable approach for expanding P450BM3‐catalyzed reactions.