Biosynthetic Incorporation of Site-Specific Isotopes in βLactam Antibiotics Enables Biophysical Studies

A biophysical understanding of the mechanistic, chemical, and physical origins underlying antibiotic action and resistance is vital to the discovery of novel therapeutics and the development of strategies to combat the growing emergence of antibiotic resistance. The site-specific introduction of stable-isotope labels into chemically complex natural products is particularly important for techniques such as NMR, IR, mass spectrometry, imaging, and kinetic isotope effects. Towards this goal, we developed a biosynthetic strategy for the site-specific incorporation of 13 C-labels into the canonical β-lactam carbonyl of penicillin G and cefotaxime, the latter via cephalosporin C. This was achieved through sulfur-replacement with 1- 13 C-L-cysteine, resulting in high isotope incorporations and mg-scale yields. Using 13 C NMR and isotope-edited IR difference spectroscopy, we illustrate how these molecules can be used to interrogate interactions with their protein targets, e.g. TEM-1 β-lactamase. This method provides a feasible route to isotopically-labeled penicillin and cephalosporin precursors for future biophysical studies.