Regio- and Stereoselective Steroid Hydroxylation by Cytochrome P450s

Steroidal C7beta alcohols and their respective esters have shown significant promise as neuroprotective and anti-inflammatory agents to treat chronic neuronal damage like stroke, brain trauma and cerebral ischemia. Since position C7 is spatially far away from any functional groups that could direct C-H activation, these transformations are not readily possible using modern synthetic organic techniques. We report P450-BM3 mutants that catalyze the oxidative hydroxylation of six different steroids with pronounced C7-regio- and beta-stereoselectivity as well as high activity. These challenging transformations were achieved by a focused mutagenesis strategy and application of a novel technology for protein library construction based on DNA assembly and USER (Uracil-Specific Excision Reagent) cloning. Upscaling reactions enabled the purification of the respective steroidal alcohols in moderate to excellent yields. The high-resolution X-ray structure and molecular dynamics simulations of the best mutant unveil the origin of regio- and stereoselectivity.

The paper by Lorenzo D’Amore, Marc Garcia-Borr s, Sílvia Osuna and co-workers appeared online recently in Angewandte Chemie-International Edition:

A. Li, C.G. Acevedo-Rocha, L. D’Amore, J. Chen, Y. Peng, M. Garcia-Borr s, C. Gao, J. Zhu, H. Rickerby, S. Osuna, J. Zhou, and M.T. Reetz
“Regio- and Stereoselective Steroid Hydroxylation at the C7-Position by Cytochrome P450 Monooxygenase Mutants”
Angew. Chem. Int. Ed. 2020, [], ASAP-
DOI: 10.1002/anie.202003139