Stereoelectronic and hydrogen-bonding effects on hydroxyproline conformation

Collagen's structural integrity and biological function derive from its characteristic triple helix, governed by a unique amino acid sequence containing the rare residue (2S,4R)-hydroxyproline (4R-Hyp). Substituting 4R-Hyp with its diastereomer 4S-Hyp markedly reduces triple-helix thermal stability and impairs collagen function. The origins of this destabilization have mostly been ascribed to stereoelectronic effects such as the gauche effect and n → π* interactions. Here, we dissect the role of different stabilizing interactions on molecular conformation using linear infrared and two-dimensional infrared spectroscopy, aided by density functional theory calculations. We investigate the structure of N-Boc-(2S,4S)-4-hydroxyproline-methyl ester (Boc-4S-Hyp-OMe) and N-Boc-(2S,4R)-4-hydroxyproline-methyl ester (Boc-4R-Hyp-OMe) in chloroform solution, a simplified model for the water-deficient environment in collagen tissues where triple helices assemble into fibrils. We find that, while stereoelectronic effects indeed influence molecular conformation, n → π* interactions only moderately alter conformational equilibria. Conversely, our results show that hydrogen bonding is pivotal: the conformation of Boc-4S-Hyp-OMe is stabilized by an intramolecular hydrogen bond, whereas Boc-4R-Hyp-OMe primarily forms intermolecular hydrogen bonds, leading to a greater intermolecular affinity. These findings highlight hydrogen bonding as a key determinant of hydroxyproline conformation at the single-residue level.