25‐Hydroxyvitamin D3 Synthesis by Enzymatic Steroid Side‐Chain Hydroxylation with Water

The hydroxylation of vitamin D₃ (VD₃, cholecalciferol) side chains to give 25‐hydroxyvitamin D₃ (25OHVD₃) is a crucial reaction in the formation of the circulating and biologically active forms of VD₃. It is usually catalyzed by cytochrome P450 monooxygenases that depend on complex electron donor systems. Cell‐free extracts and a purified Mo enzyme from a bacterium anaerobically grown with cholesterol were employed for the regioselective, ferricyanide‐dependent hydroxylation of VD₃ and proVD₃ (7‐dehydrocholesterol) into the corresponding tertiary alcohols with greater than 99 % yield. Hydroxylation of VD₃ strictly depends on a cyclodextrin‐assisted isomerization of VD₃ into preVD₃, the actual enzymatic substrate. This facile and robust method developed for 25OHVD₃ synthesis is a novel example for the concept of substrate‐engineered catalysis and offers an attractive alternative to chemical or O₂ /electron‐donor‐dependent enzymatic procedures.