Although weak interactions play subtle but important roles in dictating protein structures, their experimental detection is nontrivial. From NOE experiments we provide direct evidence for the presence of CH···π interaction, operational between the C(α)-H of the first Pro and the aromatic (Aro) side chain of Xaa, in a peptide series with the general sequence Ac-Pro-Pro-Xaa-NH(2). Indirect evidence of CH···π interaction is provided from ring current-induced upfield displacement of Pro(1) C(α)-H chemical shifts and restriction of side-chain (χ1) rotation of Xaa. A consequence of this interaction is the enhanced stability of the Pro-cisPro conformer in Ac-Pro-Pro-Xaa-NH(2) when Xaa is aromatic. The free energies associated with trans to cis transformation of the Pro-Pro moiety are 0.35, 0.59, 0.64, and 0.82 kcal/mol when Xaa is Tyr, Trp, Phe, and His (pH of 8.4), respectively. In comparison, the corresponding free energy is ∼1.55 kcal/mol when Xaa is nonaromatic. The observed population of Pro-cisPro-His and the pH-induced perturbation of electron density of the His side chain were correlated, providing further evidence for a direct role of CH···π interaction in modulating the stability of Pro-cisPro population in Ac-Pro-Pro-Aro-NH(2). Our study establishes Pro-Pro-Aro to be a new sequence motif that can stabilize Pro-cisPro peptide bonds. This study not only identifies a new structurally biased sequence motif but also directly demonstrates the role played by CH···π interactions in subtly altering conformational preferences of three-residue peptide sequences with implications on the role played by cis-peptide bonds in unfolded proteins.

Thiols can engage favorably with aromatic rings in S–H/π interactions, within abiological systems and within proteins. However, the underlying bases for S–H/π interactions are not well understood. The crystal structure of Boc-L-4-thiolphenylalanine tert-butyl ester revealed crystal organization centered on the interaction of the thiol S–H with the aromatic ring of an adjacent molecule, with a through-space Hthiol...Caromatic distance of 2.71 Å, below the 2.90 Å sum of the van der Waals radii of H and C. The nature of this interaction was further examined by DFT calculations, IR spectroscopy, solid-state NMR spectroscopy, and analysis of the Cambridge Structural Database. The S–H/π interaction was found to be driven significantly by favorable molecular orbital interactions, between an aromatic π donor orbital and the S–H σ* acceptor orbital (a π→σ* interaction). For comparison, a structural analysis of O–H/π interactions and of cation/π interactions of alkali metal cations with aromatic rings was conducted. Na+ and K+ exhibit a significant preference for the centroid of the aromatic ring and distances near the sum of the van der Waals and ionic radii, as expected for predominantly electrostatic interactions. Li+ deviates substantially from Na+ and K+. The S–H/π interaction differs from classical cation/π interactions by the preferential alignment of the S–H σ* toward the ring carbons and an aromatic π orbital rather than toward the aromatic centroid. These results describe a potentially broadly applicable approach to understanding the interactions of weakly polar bonds with π systems.

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.

The cyclic side chain of the amino acid proline confers unique conformational restraints on its backbone and side chain dihedral angles. This affects two equilibria-one at the backbone (cis/trans) and the other at the side chain (endo/exo). Substitutions on the proline ring impose additional steric and stereoelectronic effects that can further modulate both these equilibria, which in turn can also affect the backbone dihedral angle (ϕ, ψ) preferences. In this review, we have explored the conformational landscape of several termini capped mono-(2-, 3-, 4-, and 5-) substituted proline derivatives in the Cambridge Structural Database, correlating observed conformations with the nature of substituents and deciphering the underlying interactions for the observed structural biases. The impact of incorporating these derivatives within model peptides and proteins are also discussed for selected cases. Several of these substituents have been used to introduce bioorthogonal functionality and modulate structure-specific ligand recognition or used as spectroscopic probes. The incorporation of these diversely applicable functional groups, coupled with their ability to define an amino acid conformation via stereoelectronic effects, have a broad appeal among chemical biologists, molecular biophysicists, and medicinal chemists.

Compared to generic peptide bonds, the peptidyl-prolyl bond shows a strong propensity for the cis conformer. The presence of a sequence-contiguous aromatic (Aro) residue can further stabilize the cis conformer, as observed for the Aro-Pro motif. The cis propensity of the reverse sequence motif, Pro-Aro, is not so well understood, especially the effect of N-capping the Pro-Aro motif with different amino acid residues. From a comparative nuclear magnetic resonance study of two peptide series with the general sequences Ac-Xaa-Pro-Tyr-NH2 and Ac-Xaa-Pro-Ala-NH2, we present a relative thermodynamic scale that reflects how the nature of the Xaa side chain influences the cis propensity of the Xaa-Pro-Tyr motif, with Gly, Pro, and Ala at position Xaa giving the greatest enhancement of the cis-peptidyl-prolyl population. We also show that CH···π interaction between Xaa and Tyr is responsible for the enhanced cis population. However, the mere presence of the CH···π interaction does not guarantee that the peptidyl-prolyl bond will have a higher cis content in Xaa-Pro-Tyr than in Xaa-Pro-Ala. Xaa-dependent intramolecular interactions present in Xaa-trans-Pro-Tyr can nullify favorable CH···π interactions in Xaa-cis-Pro-Tyr. The relative cis-peptidyl-prolyl stabilizing propensities of Xaa (Xaa-Pro-Tyr) in proteins and in our peptide series show strong linear correlation except when Xaa is aromatic. We also explore the Xaa-Pro-Gly-Tyr sequence motif and show that mediated by a Pro-Tyr CH···π interaction, the cis-peptidyl-prolyl bond in the motif is stabilized when Xaa is Pro.

4-Substitution on prolined irectly impactsp rotein main chain conformational preferences.T he structurale ffects of N-acyl substitution and of 4-substitution were examined by NMR spectroscopy and X-ray crystallography on minimal molecules with ap roline 4S-nitrobenzoate.T he effects of N-acyl substitution on conformation werea ttenuated in the 4S-nitrobenzoate context, duet ot he minimal role of the n!p*i nteraction in stabilizing extended conformations. By X-ray crystallography,a ne xtended conformation was observedf or most molecules. The formyl derivative adopted a d conformation that is observed at the i + 2p osition of b-turns.C omputational analysis indicated that the structures observed crystallographically represent the inher-ent conformational preferences of 4S-substituted prolines with electron-withdrawing4-position substituents. The divergent conformational preferences of 4R-a nd 4S-substituted proliness uggest their wider structure-specific application in molecular design. In particular,t he proline endo ring pucker favored by 4S-substituted prolines uniquelyp romotes the d conformation [(f, y) % (À808,0 8)] found in b-turns.I nc ontrast to other acyl capping groups, the pivaloyl group strongly promoted trans amide bond and polyproline II helix conformation, with ac lose n!p*i nteraction in the crystalline state, despite the endo ring pucker,s uggesting its special capabilities in promoting compactc onformations in f due to its strongly electron-donatingc haracter.[a] N.Supporting information (including experimentalprocedures, NMR data on 3-9,N OESY spectra for 7 and 9,X -ray crystallographic data, additional analysis of computational data, 1 Ha nd 13 CNMR spectraf or all new compounds, and coordinates for all geometry-optimized structures) and the ORCID identification number(s) for the author(s) of this article can be found under:https://doi.

Phosphorylation and dephosphorylation of proteins by kinases and phosphatases are central to cellular responses and function. The structural effects of serine and threonine phosphorylation were examined in peptides and in proteins, by circular dichroism, NMR spectroscopy, bioinformatics analysis of the PDB, small-molecule X-ray crystallography, and computational investigations. Phosphorylation of both serine and threonine residues induces substantial conformational restriction in their physiologically more important dianionic forms.Threonine exhibits a particularly strong disorder-to-order transition upon phosphorylation, with dianionic phosphothreonine preferentially adopting a cyclic conformation with restricted φ (φ ~ -60˚) stabilized by three noncovalent interactions: a strong intraresidue phosphate-amide hydrogen bond, an n→π* interaction between consecutive carbonyls, and an n→σ* interaction between the phosphate Oγ lone pair and the antibonding orbital of C-Hβ that restricts the χ 2 side chain conformation. Proline is unique among the canonical amino acids for its covalent cyclization on the backbone. Phosphothreonine can mimic proline's backbone cyclization via noncovalent interactions. The preferred torsions of dianionic phosphothreonine are φ,ψ = polyproline helix or α-helix (φ ~ -60˚); χ 1 = g -; χ 2 = eclipsed C-H/O-P bonds. This structural signature is observed in diverse proteins, including the activation loops of protein kinases and protein-protein interactions. In total, these results suggest a structural basis for the differential use and evolution of threonine versus serine phosphorylation sites in proteins, with serine phosphorylation typically inducing smaller, rheostat-like changes, versus threonine phosphorylation promoting larger, step function-like switches, in proteins.

Bioorthogonal reactions allow the introduction of new functionalities into peptides, proteins, and other biological molecules. The most readily accessible amino acids for biooorthogonal reactions have modest conformational preferences or bases for molecular interactions. Herein we describe the synthesis of 4 novel amino acids containing functional groups for bioorthogonal reactions. (2S,4R)- and (2S,4S)-iodophenyl ethers of hydroxyproline are capable of modification via rapid, specific Suzuki and Sonogashira reactions in water. The synthesis of these amino acids, as Boc-, Fmoc- and free amino acids, was achieved through succinct sequences. These amino acids exhibit well-defined conformational preferences, with the 4S-iodophenyl hydroxyproline crystallographically exhibiting β-turn (ϕ, ψ ~ −80°, 0°) or relatively extended (ϕ, ψ ~ −80°, +170°) conformations, while the 4R- diastereomer prefers a more compact conformation (ϕ ~ −60°). The aryloxyproline diastereomers present the aryl groups in a highly divergent manner, suggesting their stereospecific use in molecular design, medicinal chemistry, and catalysis. Thus, the 4R- and 4S-iodophenyl hydroxyprolines can be differentially applied in distinct structural contexts. The pentynoate ester of 4R-hydroxyproline introduces an alkyne functional group within an amino acid that prefers compact conformations. The propargyl thioether of 4-thiolphenylalanine was synthesized via copper-mediated cross-coupling reaction of thioacetic acid with protected 4-iodophenylalanine, followed by thiolysis and alkylation. This amino acid combines an alkyne functional group with an aromatic amino acid and the ability to tune aromatic and side chain properties via sulfur oxidation. These amino acids provide novel loci for peptide functionalization, with greater control of conformation possible than with other amino acids containing these functional groups.