Hydrogen-Bonding Chains Twist the Peptide Bond as Revealed by Rotational Spectroscopy in N -Ethylformamide–Water Complexes

Microsolvation and hydrogen-bonding topologies critically influence local molecular geometries and electronic structures, especially in biological peptides, where solvent–peptide interactions can perturb the amide bond and affect conformational dynamics. Although peptide bonds are typically near-planar, how and to what extent local solvent molecules affect this geometry remains unresolved. In this study, we investigate stepwise microsolvation effects on the peptide bond planarity of N -ethylformamide (NEF) using rotational spectroscopy. The structures of NEF microsolvated by two to five water molecules, NEF–(H 2 O) n ( n = 2 – 5), have been determined. Conformational analysis reveals a pronounced nitrogen pyramidalization angle (θ N > 20°) in the trans-sc- NEF–(H 2 O) 3 complex, far exceeding the typical ±6° observed in proteins. The experimental nuclear quadrupole coupling constants confirm this distortion, yielding the semiexperimental value of θ N = 23.01°. This arises from nonideal hydrogen bonds in its microsolvation topology, inducing out-of-plane displacement of the amide hydrogen and thus driving a twist of its planarity. In contrast, no significant deviation is observed for other microsolvation levels, either more or less. Our results demonstrate that specific hydrogen-bonding patterns can directly perturb peptide bond planarity, highlighting the key role of local solvent effects in modulating peptide bond stability.