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Regio‐ and Stereoselective Steroid Hydroxylation at C7 by Cytochrome P450 Monooxygenase Mutants
Author(s) -
Li Aitao,
AcevedoRocha Carlos G.,
D'Amore Lorenzo,
Chen Jinfeng,
Peng Yaqin,
GarciaBorràs Marc,
Gao Chenghua,
Zhu Jinmei,
Rickerby Harry,
Osuna Sílvia,
Zhou Jiahai,
Reetz Manfred T.
Publication year - 2020
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.202003139
Subject(s) - stereoselectivity , chemistry , hydroxylation , monooxygenase , mutant , steroid , mutagenesis , cytochrome p450 , neuroprotection , combinatorial chemistry , stereochemistry , biochemistry , enzyme , gene , hormone , biology , pharmacology , catalysis
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 C7 is spatially far away from any functional groups that could direct C−H activation, these transformations are not readily accessible using modern synthetic organic techniques. Reported here are P450‐BM3 mutants that catalyze the oxidative hydroxylation of six different steroids with pronounced C7 regioselectivities and β stereoselectivities, as well as high activities. 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.