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Engineering Chemoselectivity in Hemoprotein-Catalyzed Indole Amidation

Brandenberg, Oliver F. and Miller, David C. and Markel, Ulrich and Ouald Chaib, Anissa and Arnold, Frances H. (2019) Engineering Chemoselectivity in Hemoprotein-Catalyzed Indole Amidation. ACS Catalysis, 9 (9). pp. 8271-8275. ISSN 2155-5435. PMCID PMC6959474. doi:10.1021/acscatal.9b02508.

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Here we report a cytochrome P450 variant that catalyzes C₂-amidation of 1-methylindoles with tosyl azide via nitrene transfer. Before evolutionary optimization, the enzyme exhibited two undesired side reactivities resulting in reduction of the putative iron-nitrenoid intermediate or cycloaddition between the two substrates to form triazole products. We speculated that triazole formation was a promiscuous cycloaddition activity of the P450 heme domain, while sulfonamide formation likely arose from surplus electron transfer from the reductase domain. Directed evolution involving mutagenesis of both the heme and reductase domains delivered an enzyme providing the desired indole amidation products with up to 8400 turnovers, 90% yield, and a shift in chemoselectivity from 2:19:1 to 110:12:1 in favor of nitrene transfer over reduction or triazole formation. This work expands the substrate scope of hemoprotein nitrene transferases to heterocycles and highlights the adaptability of the P450 scaffold to solve challenging chemoselectivity problems in non-natural enzymatic catalysis.

Item Type:Article
Related URLs:
URLURL TypeDescription CentralArticle
Brandenberg, Oliver F.0000-0001-5662-1234
Miller, David C.0000-0002-4560-8824
Markel, Ulrich0000-0002-5655-5342
Ouald Chaib, Anissa0000-0002-9836-7470
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2019 American Chemical Society. Received: June 14, 2019; Revised: July 27, 2019; Published: August 7, 2019. O.F.B. acknowledges support from the Deutsche Forschungsgemeinschaft (DFG grant BR 5238/1-1) and the Swiss National Science Foundation (SNF grant P300PA-171225). D.C.M. was supported by a Ruth Kirschstein NIH Postdoctoral Fellowship (F32GM128247). The authors thank Dr. Christopher K. Prier for help with initial experiments and comments on the manuscript. We also thank Dr. David K. Romney, Dr. Xiongyi Huang, and Kai Chen for helpful comments and discussions. The authors declare no competing financial interest.
Funding AgencyGrant Number
Deutsche Forschungsgemeinschaft (DFG)BR 5238/1-1
Swiss National Science Foundation (SNSF)P300PA-171225
NIH Postdoctoral FellowshipF32GM128247
Subject Keywords:biocatalysis, cytochrome P450, nitrene transfer, indole amidation, chemoselectivity
Issue or Number:9
PubMed Central ID:PMC6959474
Record Number:CaltechAUTHORS:20190807-153750847
Persistent URL:
Official Citation:Engineering Chemoselectivity in Hemoprotein-Catalyzed Indole Amidation. Oliver F. Brandenberg, David C. Miller, Ulrich Markel, Anissa Ouald Chaib, and Frances H. Arnold. ACS Catalysis 2019 9 (9), 8271-8275 DOI: 10.1021/acscatal.9b02508
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:97707
Deposited By: Tony Diaz
Deposited On:07 Aug 2019 22:44
Last Modified:17 Feb 2022 18:50

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