Site-selective Halogenation of Aromatic C-H Bonds via Electrocatalytic Flavin Adenine Dinucleotide Cofactor Regeneration
- 1Department of Chemistry
- 2Indiana University
Abstract
Flavin-dependent halogenases (FDHs) catalyze site-selective C–H halogenation of electron-rich aromatic compounds but rely on multienzyme cofactor-recycling systems to continuously supply reduced flavin (FADH 2 ). Here, we establish an overpotential-driven, mediator-assisted bioelectrocatalytic strategy for FADH 2 regeneration that eliminates auxiliary reductases, nicotinamide cofactors, and enzyme immobilization. Cyclic voltammetry confirmed the proton-dependent FAD/FADH 2 redox couple (-0.42 V vs Ag/AgCl, 3.5 M KCl) and the competing oxygen reduction reaction (ORR). Bulk electrolysis involving the FDH RebH revealed that L-ascorbic acid suppresses ORR-derived species more effectively than conventional catalase/glutathione/superoxide-dismutase systems, increasing conversion of tryptophan to 7-bromotryptophan from 20% to 46% after 24 h at -0.7 V. Increasing the cathodic potential to -1.1 V improved yields to 64% without substantially improving the faradaic efficiency (FE~ 5%). Introducing redox mediators such as 2-hydroxy-1,4-naphthoquinone (HNQ) and methyl viologen (MV 2+ ) enabled the HNQ/FAD pair to achieve quantitative halogenation within 10 h (FE ~ 14%) and the MV 2+ /FAD pair within 12 h (FE ~ 9%), enabling production of 8.8 mg of 7-bromotryptophan in a scale-up reactor. Moreover, the HNQ/FAD system exhibited a kinetic isotope effect (KIE) of ( k H / k D = 1.8) for product formation in H 2 O versus D 2 O, supporting proton involvement in productive FAD regeneration and potentially reflecting cooperation between mediator-assisted electron transfer and surface-adsorbed hydrogen (H ads ) produced during the hydrogen evolution reaction in accelerating FADH 2 formation. Extending this method to engineered RebH variant 4-V enabled halogenation of larger substrates, establishing a scalable electrochemical platform for FDH catalysis. These results establish how electron delivery, proton availability, and competing oxygen reduction govern productive electrochemical flavin regeneration and provide a scalable platform for FDH catalysis.
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BibTeX
@misc{shakir2026siteselective,
title = {{Site-selective Halogenation of Aromatic C-H Bonds via Electrocatalytic Flavin Adenine Dinucleotide Cofactor Regeneration}},
author = {Batul Shakir and Xuzhong Shen and Conor Wallace and Declan B. Mackey and Jared. C. Lewis and Ricardo Javier Vázquez},
howpublished = {ChemRxiv},
year = {2026},
month = oct,
doi = {10.26434/chemrxiv.15002776/v2},
url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15002776/v2},
note = {CC BY},
}
RIS
TY - UNPB
TI - Site-selective Halogenation of Aromatic C-H Bonds via Electrocatalytic Flavin Adenine Dinucleotide Cofactor Regeneration
AU - Batul Shakir
AU - Xuzhong Shen
AU - Conor Wallace
AU - Declan B. Mackey
AU - Jared. C. Lewis
AU - Ricardo Javier Vázquez
PY - 2026
DA - 2026/10/12
PB - ChemRxiv
DO - 10.26434/chemrxiv.15002776/v2
UR - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15002776/v2
AB - Flavin-dependent halogenases (FDHs) catalyze site-selective C–H halogenation of electron-rich aromatic compounds but rely on multienzyme cofactor-recycling systems to continuously supply reduced flavin (FADH 2 ). Here, we establish an overpotential-driven, mediator-assisted bioelectrocatalytic strategy for FADH 2 regeneration that eliminates auxiliary reductases, nicotinamide cofactors, and enzyme immobilization. Cyclic voltammetry confirmed the proton-dependent FAD/FADH 2 redox couple (-0.42 V vs Ag/AgCl, 3.5 M KCl) and the competing oxygen reduction reaction (ORR). Bulk electrolysis involving the FDH RebH revealed that L-ascorbic acid suppresses ORR-derived species more effectively than conventional catalase/glutathione/superoxide-dismutase systems, increasing conversion of tryptophan to 7-bromotryptophan from 20% to 46% after 24 h at -0.7 V. Increasing the cathodic potential to -1.1 V improved yields to 64% without substantially improving the faradaic efficiency (FE~ 5%). Introducing redox mediators such as 2-hydroxy-1,4-naphthoquinone (HNQ) and methyl viologen (MV 2+ ) enabled the HNQ/FAD pair to achieve quantitative halogenation within 10 h (FE ~ 14%) and the MV 2+ /FAD pair within 12 h (FE ~ 9%), enabling production of 8.8 mg of 7-bromotryptophan in a scale-up reactor. Moreover, the HNQ/FAD system exhibited a kinetic isotope effect (KIE) of ( k H / k D = 1.8) for product formation in H 2 O versus D 2 O, supporting proton involvement in productive FAD regeneration and potentially reflecting cooperation between mediator-assisted electron transfer and surface-adsorbed hydrogen (H ads ) produced during the hydrogen evolution reaction in accelerating FADH 2 formation. Extending this method to engineered RebH variant 4-V enabled halogenation of larger substrates, establishing a scalable electrochemical platform for FDH catalysis. These results establish how electron delivery, proton availability, and competing oxygen reduction govern productive electrochemical flavin regeneration and provide a scalable platform for FDH catalysis.
ER -