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Vibrational Stark effect (VSE) spectroscopy has become one of the most important experimental approaches to determine the strength of noncovalent, electrostatic interactions in chemistry and biology and to quantify their influence on structure and reactivity. Nitriles (C≡N) have been widely used as VSE probes, but their application has been complicated by an anomalous hydrogen bond (HB) blueshift which is not encompassed within the VSE framework. We present an empirical model describing the anomalous HB blueshift in terms of H-bonding geometry, i.e., as a function of HB distance and angle with respect to the C≡N group. This model is obtained by comparing vibrational observables from density functional theory and electrostatics from the polarizable AMOEBA force field, and it provides a physical explanation for the HB blueshift in terms of underlying multipolar and Pauli repulsion contributions. Additionally, we compare predicted blueshifts with experimental results and find our model provides a useful, direct framework to analyze HB geometry for rigid HBs, such as within proteins or chemical frameworks. In contrast, nitriles in highly dynamic H-bonding environments like protic solvents are no longer a function solely of geometry; this is a consequence of motional narrowing, which we demonstrate by simulating IR spectra. Overall, when HB geometry and dynamics are accounted for, an excellent correlation is found between observed and predicted HB blueshifts. This correlation includes different types of nitriles and HB donors, suggesting that our model is general and can aid in understanding HB blueshifts wherever nitriles can be implemented.

Molecular dynamics (MD) simulations are frequently carried out for proteins to investigate the role of electrostatics in their biological function. The choice of force field (FF) can significantly alter the MD results, as the simulated local electrostatic interactions lack benchmarking in the absence of appropriate experimental methods. We recently reported that the transition dipole moment (TDM) of the popular nitrile vibrational probe varies linearly with the environmental electric field, overcoming well-known hydrogen bonding (H-bonding) issues for the nitrile frequency and, thus, enabling the unambiguous measurement of electric fields in proteins (J. Am. Chem. Soc.2022, 144(17), 7562–7567). Herein, we utilize this new strategy to enable comparisons of experimental and simulated electric fields in protein environments. Specifically, previously determined TDM electric fields exerted onto nitrile-containing o-cyanophenylalanine residues in photoactive yellow protein are compared with MD electric fields from the fixed-charge AMBER FF and the polarizable AMOEBA FF. We observe that the electric field distributions for H-bonding nitriles are substantially affected by the choice of FF. As such, AMBER underestimates electric fields for nitriles experiencing moderate field strengths; in contrast, AMOEBA robustly recapitulates the TDM electric fields. The FF dependence of the electric fields can be partly explained by the presence of additional negative charge density along the nitrile bond axis in AMOEBA, which is due to the inclusion of higher-order multipole parameters; this, in turn, begets more head-on nitrile H-bonds. We conclude by discussing the implications of the FF dependence for the simulation of nitriles and proteins in general.

Nitriles are widely used vibrational probes; however, the interpretation of their IR frequencies is complicated by hydrogen bonding (H-bonding) in protic environments. We report a new vibrational Stark effect (VSE) that correlates the electric field projected on the −C≡N bond to the transition dipole moment and, by extension, the nitrile peak area or integrated intensity. This linear VSE applies to bothH-bonding and non-H-bonding interactions. It can therefore be generally applied to determine electric fields in all environments. Additionally, it allows for semiempirical extraction of the H-bonding contribution to the blueshift of the nitrile frequency. Nitriles were incorporated at H-bonding and non-H-bonding protein sites using amber suppression, and each nitrile variant was structurally characterized at high resolution. We exploited the combined information available from variations in frequency and integrated intensity and demonstrate that nitriles are a generally useful probe for electric fields.