期刊
ACS CATALYSIS
卷 9, 期 9, 页码 8271-8275出版社
AMER CHEMICAL SOC
DOI: 10.1021/acscatal.9b02508
关键词
biocatalysis; cytochrome P450; nitrene transfer; indole amidation; chemoselectivity
资金
- Deutsche Forschungsgemeinschaft (DFG) [BR 5238/1-1]
- Swiss National Science Foundation (SNF) [P300PA-171225]
- Ruth Kirschstein NIH Postdoctoral Fellowship [F32GM128247]
- Swiss National Science Foundation (SNF) [P300PA_171225] Funding Source: Swiss National Science Foundation (SNF)
Here we report a cytochrome P450 variant that catalyzes C-2-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.
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