Abstract
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.
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Data availability
All raw data generated and analyzed throughout this work are available from the corresponding authors upon reasonable request. The source data for Figs. 3, 4, and 5 are provided as Supplementary Data 1. Coordinate files of the DFT optimized geometries are provided as Supplementary Data 2. Additional DFT results using explicit solvent molecules, calculated absorption coefficients, fits of the linear IR spectra, and 2D-IR spectra with parallel polarization combinations are available in the Supplementary Material file attached to this paper.
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Acknowledgements
We acknowledge funding by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—SFB1551—Project No. 464588647. G. Giubertoni acknowledges that this publication is part of the project (with Project Number VI.Veni.212.240) of the research program NWO Talent Program Veni 2021, which is financed by the Dutch Research Council (NWO).
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F.M.: Conceptualization, Investigation, Formal Analysis, Writing—Original Draft Preparation, Writing—Review & Editing. P.G.A.: Formal Analysis, Writing—Review & Editing. M.B.: Conceptualization, Supervision, Writing—Review & Editing. G.G.: Conceptualization, Formal Analysis, Supervision, Writing—Review & Editing. J.H.: Conceptualization, Formal Analysis, Supervision, Writing—Review & Editing.
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Matsumura, F., Gómez Argudo, P., Bonn, M. et al. Stereoelectronic and hydrogen-bonding effects on hydroxyproline conformation. Commun Chem (2026). https://doi.org/10.1038/s42004-026-01984-x
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DOI: https://doi.org/10.1038/s42004-026-01984-x
