Regio- and Stereoselective Steroid Hydroxylation at the C7-Position by Cytochrome P450 Monooxygenase Mutants.
Mots clés
Abstrait
Steroidal C7β 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 β-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.