ZIP File: Characterisation data (such as e.g. NMR, IR, MS spectra) NMR raw data, .mnova files ChemDraw drawings (.cdx files) PDF file: Supporting information for Phenolic Substitution in Fidaxomicin: A Semisynthetic Approach to Antibiotic Activity Across Species Erik Jung,[a] Anastassia Kraimps,[a] Silvia Dittmann,[b] Tizian Griesser,[c] Jordan Costafrolaz,[d] Yves Mattenberger,[d] Simon Jurt,[a] Patrick H. Viollier,[d] Peter Sander,[c] Susanne Sievers,[b] and Karl Gademann*[a] [a] E. Jung, A. Kraimps, S. Jurt, Prof. Dr. K. Gademann Department of Chemistry, University of Zurich 8057 Zürich (Switzerland) E-mail: karl.gademann@uzh.ch [b] S. Dittmann, Dr. S. Sievers Department of Microbial Physiology and Molecular Biology Institute of Microbiology Center for Functional Genomics of Microbes University of Greifswald Greifswald (Germany) [c] T. Griesser, Prof. Dr. P. Sander Institute of Medical Microbiology University of Zurich Zurich (Switzerland) [d] J. Costafrolaz, Dr. Y. Mattenberger, Prof. Dr. P. H. Viollier Department of Microbiology and Molecular Medicine Faculty of Medicine, University of Geneva Geneva (Switzerland) Abstract of the corresponding publication: Fidaxomicin (Fdx) is a natural product antibiotic with potent activity against Clostridioides difficile and other Gram-positive bacteria such as Mycobacterium tuberculosis. Only a few Fdx derivatives have been synthesized and examined for their biological activity in the 50 years since its discovery. Fdx has a well-studied mechanism of action, namely inhibition of the bacterial RNA polymerase. Yet, the targeted organisms harbor different target protein sequences, which poses a challenge for the rational development of new semisynthetic Fdx derivatives. We introduced substituents on the two phenolic hydroxy groups of Fdx and evaluated the resulting trends in antibiotic activity against M. tuberculosis, C. difficile, and the Gram-neg- ative model organism Caulobacter crescentus. As suggested by the target protein structures, we identified the preferable derivatisation site for each organism. The derivative ortho- methyl Fdx also exhibited activity against the Gram-negative C. crescentus wild type, a first for fidaxomicin antibiotics. These insights will guide the synthesis of next-generation fidaxomicin antibiotics.