Abstract
Biocatalysis contributes significantly to the development of more sustainable synthetic pathways by using mild reaction conditions and water as a solvent. However, many relevant classes of compounds, including privileged groups in drug design, are not yet accessible via enzymatic pathways. In this context, the development of an enzymatic route to indazoles remains an unmet challenge. Here, we present a nitroreductase-triggered indazole formation, in which 2-nitrobenzylamine derivatives are converted to reactive nitrosobenzylamine intermediates that spontaneously cyclize and aromatize to indazoles. Two nitroreductases accept a series of 2-nitrobenzylamine derivatives with excellent conversions (up to >99 %). In the case of N-substituted nitrosobenzylamines, 2H-indazoles are formed, whereas other derivatives led to 1H-indazoles. The synthetic value of the nitroreductase-triggered indazole formation is further demonstrated by successful coupling with an imine reductase in a sequential cascade reaction on a 50 mg scale. With this cascade, 2H-indazoles are accessible from cheap 2-nitrobenzaldehyde and primary amines, resulting in up to 85 % conversion and 68 % isolated yield.
Data availability
Data relating to the materials and methods, experimental procedures, mechanistic studies, standard curves, response factors, enzyme kinetics, gas chromatography spectra, HRMS spectra, and NMR spectra are available in the Supplementary Information. All raw data are available from the corresponding author upon request. Protein accession codes: A0AAW3IFH5, P17117, A0AAP3FXD2, A0A7G5MPE8. Source data are provided with this paper.
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Acknowledgements
This work was supported by a NWO-VIDI grant from the Netherlands Organization for Scientific Research (NWO, VI.Vidi.213.025) and the European Research Council, ERC (grant agreement number 101075934, ReCNNSTRCT). We also acknowledge financial support from the Netherlands Organization for Scientific Research (VICI grant 724.016.002 awarded to G.J.P.). We thank Francesco G. Mutti (University of Amsterdam) for kindly providing the plasmids encoding IREDs and AspRedAm and Dirk Tischler (Ruhr-University Bochum) for kindly providing the plasmids encoding FDHs. We would also like to thank Ronald van Merkerk (University of Groningen) for developing the pH-STAT device used for mechanistic investigations.
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H.T., L.M., and E.C. designed and performed most of the experiments. A.P.L. and G.J.P. contributed NRs and knowledge about these enzymes. H.T. and S.S. conceived the project, and S.S. directed it. H.T. wrote the manuscript and all authors jointly edited the manuscript. All authors approved the final manuscript.
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Terholsen, H., Medema, L., Chernyshova, E. et al. Nitroreductase-triggered indazole formation. Nat Commun (2026). https://doi.org/10.1038/s41467-026-68926-y
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DOI: https://doi.org/10.1038/s41467-026-68926-y
