Aromatic amino acids in peptides and proteins: novel syntheses, influences on structure, and the nature of C–H/π and S–H/π interactions
Date
2016
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
In biology, the function of a protein or an enzyme is implicitly defined by its structure. The efficiency of a catalyst is dictated by the defined structure and chemical nature of the molecule. Specific functional groups within proteins and small molecules empart specialized reactivity. Functional groups can also tune and modulate noncovalent interactions in proteins and small molecules, such as salt bridges, hydrogen bonding, and aromatic interactions. Understanding how a functional group influences chemical structure and reactivity is critical for designing de novo peptide-based inhibitors, therapeutics, rationally designed proteins, catalysts, and supramolecular materials. ☐ While Nature has evolved a plethora of biological molecules with specific and reactive functional groups, the 20 natural amino acid side-chains have been limited by natural abundance and development of biosynthesis pathways. We sought to expand the “tool-box” of amino acid side-chains in proteins beyond the canonical amino acids, which provides access to side-chain groups with unique functions and reactivity. A practical methodology was developed for synthesizing peptides containing 4-thiophenylalanine, the sulfur analogue of tyrosine. 4-Thiophenylalanine was synthesized within peptides on solid-phase using a copper-mediated cross-coupling reaction. 4-Thiophenylalanine was subjected to alkylation reactions and oxidation reactions to generate a variety of different functionalized derivatives. 4-Thiophenylalanine or related derivatives can potentially be used for site-specific labeling in proteins, as spectroscopic probes for sulfur oxidation state, for studying chalogen effects in proteins and peptides, and for modulating peptide structure. ☐ In addition, 4-thiophenylalanine was synthesized as an amino acid monomer, which provided detailed insights into the fundamental nature of S–H/π aromatic interactions. The strength and geometry of the intermolecular S–H/π aromatic interaction in 4-thiophenylalanine was characterized by solution and solid-state NMR, x-ray crystallography, and IR analysis. S–H/π aromatic interactions exhibited a distinctive geometry compared to cation/π interactions in crystal structures from the Cambridge Structural Database: the S–H bond was oriented towards the edge of the face of the aromatic ring rather than the centroid. In addition, the S–H/π interaction distances were often less than the sum of the van der Waals radii for carbon and hydrogen. ab initio calculations indicated that the interaction was stabilized by a molecular orbital interaction between the aromatic π and the S–H σ* orbitals. ☐ Noncovalent interactions were explored within model peptides to gain insights into the fundamental nature of C–H/π aromatic interactions. In proteins and peptides, prolines in the cis amide bond conformation are likely to be preceded by an aromatic amino acid. It has been suggested that the aromatic-cis-proline motif is stabilized by a C–H/π aromatic interaction between proline and the aromatic ring. With practical access to a variety of non-natural aromatic amino acids, the nature of the aromatic-cis-proline C–H/π interaction was explored in more than 50 peptides of the model sequence Ac-TXPN-NH2 (where X = a substituted aromatic amino acid). Aromatic amino acids with electron-donating substituents increased the cis populations of the model peptides, with a significant upfield chemical shift in the proline Hα, suggesting a C–H/π interaction. Selected peptides Ac-TXPN-NH2 were further examined in organic solvents and characterized via van't Hoff analysis in order to understand the enthalpic and entropic contributions to aromatic-cis-proline conformations. Dipeptide motifs were further examined via solution NMR and x-ray crystallography. Extensive, C–H/π aromatic interactions with close contact distances were observed in the crystal structures of the dipeptides. The geometry and contact distances observed in the crystal structures of the aromatic-proline motifs suggested an orbital interaction between the aromatic π and the C–H * orbitals. The similar interaction geometry and contact distances between S–H/π and C–H/π aromatic interactions suggests that the π→σ*X–H interaction is general in weakly polar X–H interactions. ☐ With the unique reactivity in 4-thiophenylalanine, and the increased acidity compared to cysteine or tyrosine, we sought to utilize this aromatic amino acid in the context of native chemical ligation reactions. Native chemical ligation forms a native amide bond with peptides containing N-terminal cysteine and peptides containing C-terminal thioesters. Peptides containing 2-thiophenylalanine at the N-terminus were synthesized in a practical manner using a solid-phase cross-coupling reaction. The peptides containing 2-thiophenylalanine at the N-terminus reacted rapidly with a variety of peptides containing C-terminal thioesters. Native chemical ligation reactions mediated by 2-thiophenylalanine and were further applied for the rapid synthesis of modified proteins. Practical access to this novel amino acid provided unique insights into the mechanism of ligation reactions using aryl thiolated amino acids. ☐ With the development of practical methodologies for synthesizing a variety of aromatic amino acids within peptides, we have synthesized peptides and proteins with novel functionality and reactivity, and have explored the fundamental nature of noncovalent aromatic interactions in peptides and proteins.