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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

preprint
submitted on 21.12.2019, 04:15 and posted on 23.12.2019, 22:21 by 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.

Funding

NIH GM118044

DFG KO5464/1

History

Email Address of Submitting Author

samuel4@stanford.edu

Institution

Stanford University

Country

United States

ORCID For Submitting Author

0000-0002-5792-4015

Declaration of Conflict of Interest

Authors declare no conflict of interest

Version Notes

Original version

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