Regio‐ and Stereoselective Steroid Hydroxylation at C7 by Cytochrome P450 Monooxygenase Mutants

• Published in: Angewandte Chemie International Edition Vol. 59; no. 30; pp. 12499 - 12505
• Main Authors: Li, Aitao, Acevedo‐Rocha, Carlos G., D'Amore, Lorenzo, Chen, Jinfeng, Peng, Yaqin, Garcia‐Borràs, Marc, Gao, Chenghua, Zhu, Jinmei, Rickerby, Harry, Osuna, Sílvia, Zhou, Jiahai, Reetz, Manfred T.
• Format: Journal Article
• Language: English
• Published: WEINHEIM Wiley 20.07.2020; Wiley Subscription Services, Inc; John Wiley and Sons Inc
• Edition: International ed. in English
• Subjects: Alcohols; Brain damage; Chemistry; Chemistry, Multidisciplinary; Cloning; Cytochrome; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - genetics; Cytochrome P450; Cytochrome P450 monooxygenase; cytochromes; Cytochromes P450; Deoxyribonucleic acid; directed evolution; DNA; enzymes; Esters; Functional groups; Hydrogen Bonding; Hydroxylation; Inflammation; Ischemia; Molecular dynamics; Molecular Dynamics Simulation; Molecular structure; Mutagenesis; Mutants; Mutation; Neuroprotection; oxidation; Oxidation-Reduction; Physical Sciences; Reagents; Science & Technology; Stereoisomerism; Stereoselectivity; Steroid hormones; Steroids; Steroids - chemistry; Substrate Specificity; Traumatic brain injury; Uracil; TESTOSTERONE; oxidation; enzymes; USER CLONING; cytochromes; P450(BM3); EPIANDROSTERONE; steroids; GENE SYNTHESIS; P450; TRANSFORMATIONS; CATALYSTS; directed evolution
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.202003139

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. One mutant, six targets: The regio‐ and stereoselective C−H activation for hydroxylation of six different steroids with formation of C7β alcohols was accomplished using a single P450 mutant evolved by protein library construction based on a special DNA assembly and cloning procedure. The C7β steroidal alcohols, not readily accessible by synthetic reagents or catalysts, are of intense interest as therapeutic drugs.