The preferred conformations of peptides and proteins are dependent on local interactions that bias the conformational ensemble. The n→π* interaction between consecutive carbonyls promotes compact conformations, including the α‐helix and polyproline II helix. In order to further understand the n→π* interaction and to develop methods to promote defined conformational preferences through acyl N‐capping motifs, a series of peptides was synthesized in which the electronic and steric properties of the acyl group were modified. Using NMR spectroscopy, van't Hoff analysis of enthalpies, X‐ray crystallography, and computational investigations, we observed that more electron‐rich donor carbonyls (pivaloyl, iso‐butyryl, propionyl) promote stronger n→π* interactions and more compact conformations than acetyl or less electron‐rich donor carbonyls (methoxyacetyl, fluoroacetyl, formyl). X‐ray crystallography indicates a strong, electronically tunable preference for the α‐helix conformation, as observed directly on the φ and ψ torsion angles. Electron‐donating acyl groups promote the α‐helical conformation, even in the absence of the hydrogen bonding that stabilizes the α‐helix. In contrast, electron‐withdrawing acyl groups led to more extended conformations. More sterically demanding groups can promote trans amide bonds independent of the electronic effect on n→π* interactions. Chloroacetyl groups additionally promote n→π* interactions through the interaction of the chlorine lone pair with the proximal carbonyl π*. These data provide additional support for an important role of n→π* interactions in the conformational ensemble of disordered or unfolded proteins. Moreover, this work suggests that readily incorporated acyl N‐capping motifs that modulate n→π* interactions may be employed rationally to promote conformational biases in peptides, with potential applications in molecular design and medicinal chemistry.

Proline residues within proteins lack a traditional hydrogen bond donor. However, the hydrogens of the proline ring are all sterically accessible, with polarized CÀ H bonds at Hα and Hδ that exhibit greater partial positive character and can be utilized as alternative sites for molecular recognition. CÀ H/O interactions, between proline CÀ H bonds and oxygen lone pairs, have been previously identified as modes of recognition within protein structures and for higher-order assembly of protein structures. In order to better understand intermolecular recognition of proline residues, a series of proline derivatives was synthesized, including 4R-hydroxyproline nitrobenzoate methyl ester, acylated on the proline nitrogen with bromoacetyl and glycolyl groups, and Boc-4S-(4-iodophenyl)hydroxyproline methyl amide. All three derivatives exhibited multiple close intermolecular CÀ H/O interactions in the crystallographic state, with H•••O distances as close as 2.3 Å. These observed distances are well below the 2.72 Å sum of the van der Waals radii of H and O, and suggest that these interactions are particularly favorable. In order to generalize these results, we further analyzed the role of CÀ H/O interactions in all previously crystallized derivatives of these amino acids, and found that all 26 structures exhibited close intermolecular CÀ H/O interactions. Finally, we analyzed all proline residues in the Cambridge Structural Database of small-molecule crystal structures. We found that the majority of these structures exhibited intermolecular CÀ H/O interactions at proline CÀ H bonds, suggesting that CÀ H/O interactions are an inherent and important mode for recognition of and higher-order assembly at proline residues. Due to steric accessibility and multiple polarized CÀ H bonds, proline residues are uniquely positioned as sites for binding and recognition via CÀ H/O interactions.

Cysteine sulfonic acid (Cys-SO3H; cysteic acid) is an oxidative post-translational modification of cysteine, resulting from further oxidation from cysteine sulfinic acid (Cys-SO2H). Cysteine sulfonic acid is considered an irreversible post-translational...

Despite the importance of proline conformational equilibria (trans versus cis amide, exo versus endo ring pucker) on protein structure and function, there is a lack of convenient ways to probe proline conformation. 4,4-Difluoroproline (Dfp) was identified to be a sensitive 19F NMR-based probe of proline conformational biases and of cis-trans isomerism. Within model compounds and disordered peptides, the diastereotopic fluorines of Dfp exhibit similar chemical shifts (ΔδFF = 0–3 ppm) when a trans X–Dfp amide bond is present. In contrast, the diastereotopic fluorines exhibit a large (ΔδFF = 5–12 ppm) difference in chemical shift in a cis X–Dfp prolyl amide bond. DFT calculations, X-ray crystallography, and solid-state NMR spectroscopy indicated that the ΔδFF directly reports on the relative preference of one proline ring pucker over the other: a fluorine which is pseudo-axial (i.e. the pro-4R-F in an exo ring pucker, or the pro-4S-F in an endo ring pucker) is downfield, while a fluorine which is pseudo-equatorial (i.e. pro-4S-F when exo, or pro-4R-F when endo) is upfield. Thus, when a proline is disordered (a mixture of exo and endo ring puckers, as at trans-Pro in peptides in water), it exhibits a small Δδ. In contrast, when the Pro is ordered (i.e. when one ring pucker is strongly preferred, as in cis-Pro amide bonds, where the endo ring pucker is strongly favored), a large Δδ is observed. Dfp can be used to identify inherent induced order in peptides and to quantify proline cis-trans isomerism. Using Dfp, we discovered that the stable polyproline II helix (PPII) formed in the denatured state (8 M urea) exhibits essentially equal populations of the exo and endo proline ring puckers. In addition, the data with Dfp suggested the specific stabilization of PPII by water over other polar solvents. These data strongly support the importance of carbonyl solvation and n→π* interactions for the stabilization of PPII. Dfp was also employed to quantify proline cis-trans isomerism as a function of phosphorylation and the R406W mutation in peptides derived from the intrinsically disordered protein tau. Dfp is minimally sterically disruptive and can be incorporated in expressed proteins, suggesting its broad application in understanding proline cis-trans isomerization, protein folding, and local order in intrinsically disordered proteins.