Collagen Stability: Insights from NMR Spectroscopic and Hybrid Density Functional Computational Investigations of the Effect of Electronegative Substituents on Prolyl Ring Conformations

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Abstract
Collagen-like peptides of the type (Pro-Pro-Gly)10 fold into stable triple helices. An electron-withdrawing substituent at the Hγ3 ring position of the second proline residue stabilizes these triple helices. The aim of this study was to reveal the structural and energetic origins of this effect. The approach was to obtain experimental NMR data on model systems and to use these results to validate computational chemical analyses of these systems. The most striking effects of an electron-withdrawing substituent are on the ring pucker of the substituted proline (Proi) and on the trans/cis ratio of the Xaai-1−Proi peptide bond. NMR experiments demonstrated that N-acetylproline methyl ester (AcProOMe) exists in both the Cγ-endo and Cγ-exo conformations (with the endo conformation slightly preferred), N-acetyl-4(R)-fluoroproline methyl ester (Ac-4R-FlpOMe) exists almost exclusively in the Cγ-exo conformation, and N-acetyl-4(S)-fluoroproline methyl ester (Ac-4S-FlpOMe) exists almost exclusively in the Cγ-endo conformation. In dioxane, the Ktrans/cis values for AcProOMe, Ac-4R-FlpOMe, and Ac-4S-FlpOMe are 3.0, 4.0, and 1.2, respectively. Density functional theory (DFT) calculations with the (hybrid) B3LYP method were in good agreement with the experimental data. Computational analysis with the natural bond orbital (NBO) paradigm shows that the pucker preference of the substituted prolyl ring is due to the gauche effect. The backbone torsional angles, φ and ψ, were shown to correlate with ring pucker, which in turn correlates with the known φ and ψ angles in collagen-like peptides. The difference in Ktrans/cis between AcProOMe and Ac-4R-FlpOMe is due to an n → π* interaction associated with the Bürgi−Dunitz trajectory. The decrease in Ktrans/cis for Ac-4S-FlpOMe can be explained by destabilization of the trans isomer because of unfavorable electronic and steric interactions. Analysis of the results herein along with the structures of collagen-like peptides has led to a theory that links collagen stability to the interplay between the pyrrolidine ring pucker, φ and ψ torsional angles, and peptide bond trans/cis ratio of substituted proline residues.

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