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13C_MS_SI_ChemRxiv_Final.pdf (1.57 MB)
Biosynthetic Incorporation of Site-Specific Isotopes in βLactam Antibiotics Enables Biophysical Studies
Preprints are manuscripts made publicly available before they have been submitted for formal peer review and publication. They might contain new research findings or data. Preprints can be a draft or final version of an author's research but must not have been accepted for publication at the time of submission.
submitted on 21.12.2019 and posted on 23.12.2019by Jacek Kozuch, Samuel Schneider, Steven Boxer
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 13C-labels into the canonical
β-lactam carbonyl of penicillin G and cefotaxime, the latter via cephalosporin
C. This was achieved through sulfur-replacement with 1-13C-L-cysteine,
resulting in high isotope incorporations and mg-scale yields. Using 13C
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.