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PreprintCited by 44Enzyme Catalysis and ImmobilizationVanadium and Halogenation ChemistryOxidative Organic Chemistry Reactions

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The Single Component Flavin Reductase/Flavin Dependent Halogenase AetF is a Versatile Catalyst for Selective Bromination and Iodination of Arenes and Olefins

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Jiang, Yuhua, Snodgrass, Harrison M., Zubi, Yasmine S., Roof, Caitlin V., Guan, Yanfei, Mondal, Dibyendu, Honeycutt, Nicholas H., Lee, Johnny W., Lewis, Russell D., Martinez, Carlos, Lewis, Jared C.·2022·Angewandte Chemie International Edition
DOI 10.26434/chemrxiv-2022-88xzvPMC PMC9772203

Preprint — published as 10.1002/anie.202214610 · Paper page

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Abstract

Flavin-dependent halogenases (FDHs) natively catalyze selective halogenation of electron rich aromatic and enolate groups. Nearly all FDHs reported to date require a separate flavin reductase to supply the FADH2¬ required by these enzymes. This requirement complicates biocatalysis applications since flavin reductases are not widely available, they add to the protein waste that must be removed during product isolation, and they can lead to undesired background reactions. In this study, we establish that the single component flavin reductase/flavin dependent halogenase AetF catalyzes halogenation of a diverse set of substrates. High site selectivity was observed in many cases, and activity on relatively unactivated substrates and heterocyclic substrates was demonstrated. High enantioselectivity was observed for atroposelective halogenation and halocyclization reactions. Site-selective iodination and enantioselective cycloiodoetherification was also possible using AetF. The substrate and reaction scope of AetF, along with the fact that this enzyme requires only a commercially available glucose dehydrogenase to drive its halogenase activity, suggest that it has the potential to greatly improve the utility of biocatalytic halogenation.

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Figure 1 A) Halogenation of electron rich aromatic compounds has been reported using either two‐component or fused flavin reductase/halogenase systems like RebH/RebF. [4] B) Native products of the two known single component flavin reductase/halogenases (R/H), Bmp5 and AetF.[ 17 , 18 ]
Figure 1 A) Halogenation of electron rich aromatic compounds has been reported using either two‐component or fused flavin reductase/halogenase systems like RebH/RebF. [4] B) Native products of the two known single component flavin reductase/halogenases (R/H), Bmp5 and AetF.[ 17 , 18 ]CC BY-NC-ND · PubMed Central
Figure 2 Representative examples of substrates brominated by AetF (see also Figure S1 and Table S1). Conversions were determined from relative integration values for starting material and product for reactions conducted on 75 μL scale; selected isolated yields (isol.) and assay yields (AY) relative to authentic internal standards were also obtained for reactions conducted on 20–30 mL and 75 μL scale, respectively. Halenium affinity (HalA) values [25] were calculated as previously reported. [20] GDH=glucose dehydrogenase, Cat.=catalase.
Figure 2 Representative examples of substrates brominated by AetF (see also Figure S1 and Table S1). Conversions were determined from relative integration values for starting material and product for reactions conducted on 75 μL scale; selected isolated yields (isol.) and assay yields (AY) relative to authentic internal standards were also obtained for reactions conducted on 20–30 mL and 75 μL scale, respectively. Halenium affinity (HalA) values [25] were calculated as previously reported. [20] GDH=glucose dehydrogenase, Cat.=catalase.CC BY-NC-ND · PubMed Central
Figure 3 A) AetF‐catalyzed aromatic iodination and cycloiodoetherification. Conversions were determined from relative integration values for starting material and product for reactions conducted on 75 μL scale; selected isolated yields (isol.) and assay yields (AY) relative to authentic internal standards were also obtained for reactions conducted on 20–30 mL scale. B) Bromination and iodination of tryptophan catalyzed by AetF (red and orange traces, respectively) and AetF K258A (blue traces), showing no conversion to either halogenated product using the latter enzyme. GDH=glucose dehydrogenase, Cat.=catalase.
Figure 3 A) AetF‐catalyzed aromatic iodination and cycloiodoetherification. Conversions were determined from relative integration values for starting material and product for reactions conducted on 75 μL scale; selected isolated yields (isol.) and assay yields (AY) relative to authentic internal standards were also obtained for reactions conducted on 20–30 mL scale. B) Bromination and iodination of tryptophan catalyzed by AetF (red and orange traces, respectively) and AetF K258A (blue traces), showing no conversion to either halogenated product using the latter enzyme. GDH=glucose dehydrogenase, Cat.=catalase.CC BY-NC-ND · PubMed Central
Figure 4 Overlays of A) a AetF⋅FAD⋅NADP model constructed using AlphaFold with cofactors docked in using Rosetta Ligand (green) with the crystal structure of a predicted ancestral FPMO (PDB ID 6SF0, grey) [32] and B) the AetF⋅FAD⋅Trp model active site (green residues) with K79 and bound Trp substrate from the FDH RebH (PDB ID 2OA1, grey residues). [34]
Figure 4 Overlays of A) a AetF⋅FAD⋅NADP model constructed using AlphaFold with cofactors docked in using Rosetta Ligand (green) with the crystal structure of a predicted ancestral FPMO (PDB ID 6SF0, grey) [32] and B) the AetF⋅FAD⋅Trp model active site (green residues) with K79 and bound Trp substrate from the FDH RebH (PDB ID 2OA1, grey residues). [34]CC BY-NC-ND · PubMed Central

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Authors
Jiang, Y., Snodgrass, H. M., Zubi, Y. S., Roof, C. V., Guan, Y., Mondal, D., Honeycutt, N. H., Lee, J. W., Lewis, R. D., Martinez, C., & Lewis, J. C.
Affiliations
  • Indiana University Bloomington (US)
  • Pfizer (United States) (US)
Corresponding author
Lewis, Jared C.
ORCID
Venue
Angewandte Chemie International Edition
Publisher
American Chemical Society (ACS)
Published
2022-10-05
Volume
61
Issue
51
Pages
e202214610-e202214610
ISSN
1433-7851 (ISSN-L)
DOI
10.26434/chemrxiv-2022-88xzv
PMC
PMC9772203
OpenAlex
W4307230595
Cited by
44
References
1
Citations by year
2026: 6 · 2025: 14 · 2024: 15 · 2023: 8 · 2022: 1
FWCI
3.10
Type
journal article
Language
en
Open access
Licence
Locations
  • Angewandte Chemie International Edition (publishedVersion, cc-by-nc-nd)
  • ChemRxiv (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
National Institutes of Health (R01 GM115665)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
  • Physical Sciences › Chemistry › Inorganic Chemistry › Vanadium and Halogenation Chemistry
  • Physical Sciences › Chemistry › Organic Chemistry › Oxidative Organic Chemistry Reactions
Keywords
flavin-dependent halogenases, flavin reductase, selective bromination, selective iodination, glucose dehydrogenase, bromolactonization
MeSH terms
Alkenes, Flavins, Oxidoreductases, Halogenation, Biocatalysis

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