Directed evolution of P411 enzymes for amination of inert C-H bonds
Copyright © 2023. Published by Elsevier Inc..
Functionalizing inert C-H bonds selectively is a formidable task due to their strong bond energy and the difficulty of distinguishing chemically similar C-H bonds. While enzymatic oxygenation of C-H bonds is ubiquitous and well established, there is currently no known natural enzymatic process for direct nitrogen insertion. Instead, nature typically relies on pre-oxidized compounds for nitrogen incorporation. Direct biocatalytic C-H amination methods developed in the last few years are only selective for activated C-H bonds that contain specific groups such as benzylic, allylic, or propargylic groups. However, we recently used directed evolution to generate cytochrome P411 enzymes (engineered P450 enzymes with axial ligand mutation from cysteine to serine) that directly aminate inert C-H bonds with high site-, diastereo-, and enantioselectivity. Using these enzymes, we demonstrated the regiodivergent desymmetrization of methylcyclohexane, among other reactions. This chapter provides a comprehensive account of the experimental protocols used to evolve P411s for aminating unactivated C-H bonds. These methods are illustrative and can be adapted for other directed enzyme evolution campaigns.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:693 |
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| Enthalten in: |
Methods in enzymology - 693(2023) vom: 03., Seite 1-30 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Das, Anuvab [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 27.11.2023 Date Revised 27.11.2023 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/bs.mie.2023.09.009 |
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| Förderinstitution / Projekttitel: |
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| 520 | |a Copyright © 2023. Published by Elsevier Inc. | ||
| 520 | |a Functionalizing inert C-H bonds selectively is a formidable task due to their strong bond energy and the difficulty of distinguishing chemically similar C-H bonds. While enzymatic oxygenation of C-H bonds is ubiquitous and well established, there is currently no known natural enzymatic process for direct nitrogen insertion. Instead, nature typically relies on pre-oxidized compounds for nitrogen incorporation. Direct biocatalytic C-H amination methods developed in the last few years are only selective for activated C-H bonds that contain specific groups such as benzylic, allylic, or propargylic groups. However, we recently used directed evolution to generate cytochrome P411 enzymes (engineered P450 enzymes with axial ligand mutation from cysteine to serine) that directly aminate inert C-H bonds with high site-, diastereo-, and enantioselectivity. Using these enzymes, we demonstrated the regiodivergent desymmetrization of methylcyclohexane, among other reactions. This chapter provides a comprehensive account of the experimental protocols used to evolve P411s for aminating unactivated C-H bonds. These methods are illustrative and can be adapted for other directed enzyme evolution campaigns | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Amination | |
| 650 | 4 | |a Biocatalysis | |
| 650 | 4 | |a Cytochrome P411 | |
| 650 | 4 | |a C–H functionalization | |
| 650 | 4 | |a Directed evolution | |
| 650 | 4 | |a Enzyme engineering | |
| 650 | 4 | |a Nitrene transfer | |
| 650 | 4 | |a Screening | |
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| 700 | 1 | |a Athavale, Soumitra V |e verfasserin |4 aut | |
| 700 | 1 | |a Alfonzo, Edwin |e verfasserin |4 aut | |
| 700 | 1 | |a Long, Yueming |e verfasserin |4 aut | |
| 700 | 1 | |a Arnold, Frances H |e verfasserin |4 aut | |
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