Biocatalytic C14-Hydroxylation on Androstenedione Enabled Modular Synthesis of Cardiotonic Steroids

Cardiotonic steroids are a unique class of natural products, possessing a steroid framework with a characteristic hydroxyl group at C14 and a β-oriented five- or six-membered unsaturated lactone substituent at C17. Installation of a hydroxyl group at the C14 position by direct C–H hydroxylation is proved to be challenging through chemical transformation. Herein, we report the identification of two P450 enzymes, CYP11411 and CYP44476, each of which can convert androstenedione (AD) to 14α-OH-AD directly, from the plant Calotropis gigantea and the toad Bufo gargarizans, respectively. The obtained 14α-OH-AD can then be chemically converted to a key 14β-OH steroid intermediate, which serves as a basis for the total synthesis of cardiotonic steroids including bufotalin, bufogenin B, and digitoxigenin by the modular installation of a five- or six-membered lactone ring at the C17 position.