Stereoselective synthesis of allopregnanolone catalyzed by ketoreductase

The U.S. Food and Drug Administration (FDA) has approved a synthetic version of allopregnanolone, called brexanolone (brand name Zulresso), for the treatment of postpartum depression. We found that the NADPH-dependent short-chain dehydrogenase LfSDR1 can stereoselectively catalyze the reduction of 5α-dihydroprogesterone to allopregnanolone. Our studies show that a series of variants of LfSDR1 with single mutation such as E141L and I192V have excellent diastereoselectivity (3R:3S >99:1) and regioselectivity, and that the combined mutation E141L/I192V has significantly increased reactivity in the reduction of dihydroprogesterone while retaining the original single mutation selectivity. The use of a glucose dehydrogenase (GDH)-based cofactor cycling system, combined with the addition of co-solvents and solubilisers to increase the solubility of steroid substrates, significantly improved the reduction reaction. Finally, up to 20 mM (6.5 mg/mL) of 5α-dihydroprogesterone encapsulated by 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) can be converted to allopregnanolone within 3 h (>90% conversion). This study provides an effective method for the highly selective, low-cost biopreparation of sterols.