Diverse engineered heme proteins enable stereodivergent cyclopropanation of unactivated alkenes
Abstract
Developing catalysts that produce each stereoisomer of a desired product selectively is a longstanding synthetic challenge. Biochemists have addressed this challenge by screening nature's diversity to discover enzymes that catalyze the formation of complementary stereoisomers. We show here that the same approach can be applied to a new-to-nature enzymatic reaction, alkene cyclopropanation via carbene transfer. By screening diverse native and engineered heme proteins, we identified globins and serine-ligated "P411" variants of cytochromes P450 with promiscuous activity for cyclopropanation of unactivated alkene substrates. We then enhanced their activities and stereoselectivities by directed evolution: just 1–3 rounds of site-saturation mutagenesis and screening generated enzymes that transform unactivated alkenes and electron-deficient alkenes into each of the four stereoisomeric cyclopropanes with up to 5,400 total turnovers and 98% enantiomeric excess. These fully genetically encoded biocatalysts function in whole Escherichia coli cells in mild, aqueous conditions and provide the first example of enantioselective, intermolecular iron-catalyzed cyclopropanation of unactivated alkenes.
Additional Information
© 2018 American Chemical Society. ACS AuthorChoice - This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Received: November 10, 2017; Publication Date (Web): February 21, 2018. This work was supported by the National Science Foundation Division of Molecular and Cellular Biosciences (Grant MCB-1513007) and the Office of Chemical, Bioengineering, Environmental and Transport Systems SusChEM Initiative (Grant CBET-1403077). The authors thank Dr. Nathan Dalleska, Aurapat Ngamnithiporn, and Dr. Scott C. Virgil for analytical chiral GC support, and Dr. Stephan C. Hammer and Dr. Xiongyi Huang for helpful discussions and critical reading of the manuscript. A.M.K. gratefully acknowledges support from Caltech's Center for Environmental Microbial Interactions and the NSF Graduate Research Fellowship (Grant No. DGE-1745301). R.D.L. is supported by an NIH–National Research Service Award training grant (5 T32 GM07616). O.F.B. acknowledges support from the Deutsche Forschungsgemeinschaft (Grant No. BR 5238/1-1) and the Swiss National Science Foundation (Grant No. P300PA-171225). A provisional patent application has been filed through the California Institute of Technology based on the results presented here. The authors declare no competing financial interest.Attached Files
Published - acscentsci.7b00548.pdf
Submitted - 20171219_Knight_ChemRxiv_submission.pdf
Supplemental Material - oc7b00548_si_001.pdf
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Additional details
- PMCID
- PMC5879470
- Eprint ID
- 83972
- Resolver ID
- CaltechAUTHORS:20171220-092532762
- NSF
- MCB-1513007
- NSF
- CBET-1403077
- Caltech Center for Environmental Microbial Interactions (CEMI)
- NSF Graduate Research Fellowship
- DGE-1745301
- NIH Predoctoral Fellowship
- 5 T32 GM07616
- Deutsche Forschungsgemeinschaft (DFG)
- BR 5238/1-1
- Swiss National Science Foundation (SNSF)
- P300PA-171225
- Created
-
2017-12-20Created from EPrint's datestamp field
- Updated
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2022-03-17Created from EPrint's last_modified field
- Caltech groups
- Caltech Center for Environmental Microbial Interactions (CEMI), Rosen Bioengineering Center