25‐Hydroxyvitamin D 3 Synthesis by Enzymatic Steroid Side‐Chain Hydroxylation with Water

The hydroxylation of vitamin D 3 (VD 3 , cholecalciferol) side chains to give 25‐hydroxyvitamin D 3 (25OHVD 3 ) is a crucial reaction in the formation of the circulating and biologically active forms of VD 3 . 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 VD 3 and proVD 3 (7‐dehydrocholesterol) into the corresponding tertiary alcohols with greater than 99 % yield. Hydroxylation of VD 3 strictly depends on a cyclodextrin‐assisted isomerization of VD 3 into preVD 3 , the actual enzymatic substrate. This facile and robust method developed for 25OHVD 3 synthesis is a novel example for the concept of substrate‐engineered catalysis and offers an attractive alternative to chemical or O 2  /electron‐donor‐dependent enzymatic procedures.