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

Noncovalent interactions underlie nearly all molecular processes in the condensed phase from solvation to 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 toward 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 electric fields are correlated to conformation-dependent effects of the flexible PenG side chain, including both the local solvation structure and solute conformational sampling in MD. Additionally, we identified that MD-based electric fields are consistently overestimated in three-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.