A Green Chemoenzymatic Route to 28-epi-Homobrassinolide Enabled by OsO₄-Free Strategy and Engineered Baeyer-Villiger...

28-epi-homobrassinolide (28-epi-HBL), a bioactive analog of the steroidal phytohormone brassinolide, exhibits broad biological activities and considerable application potential. However, conventional chemical routes remain limited by tetrahydroxylation step that relies on highly toxic osmium tetroxide in combination with costly chiral ligands, whereas the Baeyer-Villiger oxidation step requires strong oxidants and often exhibits limited regioselective control. In this work, optimization of tetrahydroxylation was achieved using the Shing-Plietker reaction, thereby avoiding the use of osmium tetroxide. Moreover, an engineered Baeyer-Villiger monooxygenase (CpnB_BL) was heterologously expressed and used for regioselective B-ring lactonization under mild conditions. A ZIF-8-based immobilization strategy was established for this engineered enzyme, further improving its stability and solvent tolerance, and increasing the isolated yield of the Baeyer-Villiger step to 83%. These optimized strategies were subsequently applied to the total synthesis of 28-epi-HBL, achieving an overall yield of 44.9%. This represents a marked improvement over the highest previously reported yield in the literature, which stands at approximately 20%. Collectively, this study lays the groundwork for the development of more precise and sustainable synthetic routes to 28-epi-HBL.