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

Abstract

‡ These authors contributed equally to this work.Abstract: 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 sitespecific 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.

The Interplay of Electrostatics and Chemical Positioning in the Evolution of Antibiotic Resistance in TEM β-Lactamases

“…We utilized isotope-edited 12 C– 13 C difference spectroscopy to determine the active site electric fields using the VSE, made possible by site-specific isotope incorporation of 13 C into common β-lactam antibiotics . This method enables detection of a single CO stretching frequency even amid the extensive protein backbone signal (Figures S6,7).…”

“…We utilized isotope-edited 12 C− 13 C difference spectroscopy to determine the active site electric fields using the VSE, made possible by site-specific isotope incorporation of 13 C into common β-lactam antibiotics. 34 This method enables detection of a single CO stretching frequency even amid the extensive protein backbone signal (Figures S6,7). 35 In order to trap the ES and AE species for the intrinsically slow IR measurements, we introduce the S70G and E166N mutations, respectively, to monitor the mechanistic intermediates.…”

Testing the Limitations of MD-Based Local Electric Fields Using the Vibrational Stark Effect in Solution: Penicillin G as a Test Case

“…These results establish methods for extending the VSE to complex (bio)molecules to quantify environment-dependent electrostatic forces, e.g., upon binding of PenG to its targets the penicillin-binding proteins or β-lactamases. 90…”

The Interplay of Electrostatics and Chemical Positioning in the Evolution of Antibiotic Resistance in TEM β-Lactamases

“…through averaging 256-512 scans from 4000-1000 cm -1 with 1 cm -1 resolution after 600 secs of dry air purging. 34 All protein-substrate measurements were performed similarly with a final concentration of ca. 2.3 mM TEM and a slight excess of substrate (ca.…”

“…We utilized isotope-edited 12 C -13 C difference spectroscopy to determine the active site electric fields using the VSE, made possible by site-specific isotope incorporation of 13 C into common β-lactam antibiotics. 34 This method enables detection of a single C=O stretching frequency even amid the extensive protein backbone signal (Figure S5 & 7). 35 In order to trap the ES and AE species for the intrinsically slow IR measurements, we introduce the S70G and E166N mutations, respectively, to monitor the mechanistic intermediates.…”