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Discovery of a fluorinated macrobicyclic antibiotic through chemical synthesis

Nature Chemistry volume 17, pages 582–589 (2025)Cite this article

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Abstract

The emergence of bacterial antimicrobial resistance threatens to undermine the utility of antibiotic therapy in medicine. This threat can be addressed, in part, by reinventing existing antibiotic classes using chemical synthesis. Here we present the discovery of BT-33, a fluorinated macrobicyclic oxepanoprolinamide antibiotic with broad-spectrum activity against multidrug-resistant bacterial pathogens. Structure–activity relationships within the macrobicyclic substructure reveal structural features that are essential to the enhanced potency of BT-33 as well as its increased metabolic stability relative to its predecessors clindamycin, iboxamycin and cresomycin. Using X-ray crystallography, we determine the structure of BT-33 in complex with the bacterial ribosome revealing that its fluorine atom makes an additional van der Waals contact with nucleobase G2505. Through variable-temperature 1H NMR experiments, density functional theory calculations and vibrational circular dichroism spectroscopy, we compare macrobicyclic homologues of BT-33 and a C7 desmethyl analogue and find that the C7 methyl group of BT-33 rigidifies the macrocyclic ring in a conformation that is highly preorganized for ribosomal binding.

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Fig. 1: Synthesis of diversifiable intermediate 1.
Fig. 2: Discovery synthesis of macrobicyclic OPP BT-33.
Fig. 3: MIC values of selected macrobicyclic OPP analogues illustrate structure–activity relationships.
Fig. 4: In vitro antibacterial activity and in vivo pharmacokinetics of BT-33.
Fig. 5: Structure of BT-33 in complex with the bacterial ribosome.
Fig. 6: Conformational analyses of macrobicyclic acetamides 11 and 12.

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Data availability

All data supporting this study are made available in the main text or the Supplementary Information. Crystallographic data for the structure of intermediate 8 were deposited in the Cambridge Crystallographic Data Centre (deposition number 2376105) and are accessible free of charge. Coordinates and structure factors were deposited in the RCSB PDB for the wild-type T. thermophilus 70S ribosome in complex with mRNA, deacylated A-site tRNAPhe, aminoacylated P-site fMet-tRNAiMet, deacylated E-site tRNAPhe and BT-33 with accession code 9D0J. Reference structures used for analysis can be found in the RCSB PDB with accession codes 4V7V and 6XHW. Source data are provided with this paper.

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Acknowledgements

We gratefully acknowledge M. J. Mitcheltree and G. Testolin for the design conception and first synthesis of (nine-membered) macrobicyclic thiolincosamines; S. L. Zheng for the collection of X-ray crystallographic data for intermediate 8 under the support of the Major Research Instrumentation (MRI) Program of the National Science Foundation (NSF), award 2216066; D. Cui for the support of variable-temperature 1H NMR experiments; J. X. Wang for the collection of mass spectrometry data; M. D. Cameron for the collection of liver microsomal stability data; Pharmaron (Ningbo, China) for collection of murine pharmacokinetic data; and D. R. Andes, L. Burrows, S. Chiang, P. Courvalin, T. J. Dougherty, R. Duggal, M. S. Gilmore, C. Grillot-Courvalin, T. Grossman, V. Hauryliuk, C. Keith, S. Lahiri, F. H. Lebreton, A. S. Mankin, H. E. Moser, D. P. Nicolau, K. M. Otte, A. Pisipati, S. Projan, N. C. Vazquez-Laslop and B. Wohl for their invaluable insights over the course of our research.

This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, grants R01-AI168228 (A.G.M.) and R21-AI163466 (Y.S.P.); National Institute of General Medical Sciences, grants R35-GM151957 (Y.S.P.) and R01-GM132302 (Y.S.P.); Illinois State Startup Funds (Y.S.P.); and Corning Fund for Faculty Development (R.Y.L.).

This work is based upon research conducted at the Northeastern Collaborative Access Team (NE-CAT) beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (NIH; grant P30-GM124165 to NE-CAT). The Eiger 16M detector on the 24-ID-E beamline is funded by an NIH Office of Research Infrastructure Programs (ORIP) High-End Instrumentation (HEI) grant (grant S10-OD021527 to NE-CAT). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357.

CARB-X funding for this research (A.G.M.) is supported by federal funds from the US Department of Health and Human Services (HHS), Administration for Strategic Preparedness and Response, Biomedical Advanced Research and Development Authority under agreement number 75A50122C00028, and by awards from Wellcome (WT224842), Germany’s Federal Ministry of Education and Research (BMBF) and the UK Department of Health and Social Care as part of the Global Antimicrobial Resistance Innovation Fund (GAMRIF). The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of CARB-X or its funders.

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Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    Ben I. C. Tresco, Kelvin J. Y. Wu, Antonio Ramkissoon, Michael Purdy, Dominic N. Y. See, Richard Y. Liu & Andrew G. Myers

  2. Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL, USA

    Elena V. Aleksandrova & Yury S. Polikanov

  3. Department of Pharmaceutical Sciences, University of Illinois at Chicago, Chicago, IL, USA

    Yury S. Polikanov

  4. Center for Biomolecular Sciences, University of Illinois at Chicago, Chicago, IL, USA

    Yury S. Polikanov

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  1. Ben I. C. Tresco
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Contributions

B.I.C.T. designed, synthesized and characterized BT-33. B.I.C.T., K.J.Y.W. and D.N.Y.S. synthesized and characterized macrobicyclic oxepanoprolinamide analogues. A.R., B.I.C.T. and K.J.Y.W. designed and performed antimicrobial susceptibility testing assays for all analogues. E.V.A. and Y.S.P. designed and performed X-ray crystallography experiments. M.P. performed VCD measurements. R.Y.L. performed DFT calculations. A.G.M., Y.S.P. and R.Y.L. supervised the experiments. B.I.C.T. and A.G.M. wrote the manuscript. All authors interpreted the results and commented on the manuscript.

Corresponding authors

Correspondence to Richard Y. Liu, Yury S. Polikanov or Andrew G. Myers.

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Competing interests

B.I.C.T., K.J.Y.W. and A.G.M. have filed international patent application WO/2023/205206, ‘Lincosamides and Uses Thereof’, and US provisional patent application US/63/676,017, ‘Synthesis of macrobicyclic Thiolincosamines’. B.I.C.T., K.J.Y.W. and A.G.M. are shareholders of Kinvard Bio, Inc. All other authors declare no competing interests.

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Nature Chemistry thanks Dale Boger and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–7, Tables 1–5, synthetic methods and characterization data for new OPP analogues and coordinates and energies for DFT-optimized structures.

Reporting Summary

Supplementary Video 1

Visualization of BT-33 binding to the T. thermophilus ribosome.

Supplementary Data 1

The CIF file for intermediate 8.

Source data

Source Data Fig. 4

MIC data for Fig. 4a,b and plasma concentrations for single-dose pharmacokinetics study.

Source Data Fig. 6

Calculated and experimental VCD spectra of 12.

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Tresco, B.I.C., Wu, K.J.Y., Ramkissoon, A. et al. Discovery of a fluorinated macrobicyclic antibiotic through chemical synthesis. Nat. Chem. 17, 582–589 (2025). https://doi.org/10.1038/s41557-025-01738-7

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