<div>Non-covalent interactions underlie nearly all molecular processes in the condensed phase from solvation to</div><div>catalysis. Their quantification within a physically consistent framework remains challenging. Experimental vibrational Stark effect (VSE)-based solvatochromism can be combined with molecular dynamics (MD) simulations to quantify the electrostatic forces in solute-solvent interactions for small rigid molecules and, by extension, when these solutes bind in enzyme active sites. While generalizing this approach towards more complex (bio)molecules, such as the conformationally flexible and charged penicillin G (PenG), we were surprised to observe inconsistencies in MD-based electric fields. Combining synthesis, VSE spectroscopy, and computational methods, we provide an intimate view on the origins of these discrepancies. We observe that the electrics fields are correlated to conformation-dependent effects of the flexible PenG side-chain, including both local solvation structure and solute conformational sampling in MD. Additionally, we identified that MD-based electric fields are consistently overestimated in 3-point water models in the vicinity of charged groups; this cannot be entirely ameliorated using polarizable force fields (AMOEBA) or advanced water models. This work demonstrates the value of the VSE as a direct method for experiment-guided refinements of MD force fields and establishes a general reductionist approach to calibrating vibrational probes for complex (bio)molecules.</div>

<div>Non-covalent interactions underlie nearly all molecular processes in the condensed phase from solvation to</div><div>catalysis. Their quantification within a physically consistent framework remains challenging. Experimental vibrational Stark effect (VSE)-based solvatochromism can be combined with molecular dynamics (MD) simulations to quantify the electrostatic forces in solute-solvent interactions for small rigid molecules and, by extension, when these solutes bind in enzyme active sites. While generalizing this approach towards more complex (bio)molecules, such as the conformationally flexible and charged penicillin G (PenG), we were surprised to observe inconsistencies in MD-based electric fields. Combining synthesis, VSE spectroscopy, and computational methods, we provide an intimate view on the origins of these discrepancies. We observe that the electrics fields are correlated to conformation-dependent effects of the flexible PenG side-chain, including both local solvation structure and solute conformational sampling in MD. Additionally, we identified that MD-based electric fields are consistently overestimated in 3-point water models in the vicinity of charged groups; this cannot be entirely ameliorated using polarizable force fields (AMOEBA) or advanced water models. This work demonstrates the value of the VSE as a direct method for experiment-guided refinements of MD force fields and establishes a general reductionist approach to calibrating vibrational probes for complex (bio)molecules.</div>

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.

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.