Peptide recognition by a synthetic receptor at subnanomolar concentrations.

This paper describes the discovery and characterization of a dipeptide sequence, Lys-Phe, that binds to the synthetic receptor cucurbit[8]uril (Q8) in neutral aqueous solution with subnanomolar affinity when located at the N-terminus. The thermodynamic and structural basis for the binding of Q8 to a series of four pentapeptides was characterized by isothermal titration calorimetry, NMR spectroscopy, and X-ray crystallography. Submicromolar binding affinity was observed for the peptides Phe-Lys-Gly-Gly-Tyr (FKGGY, 0.3 µM) and Tyr-Leu-Gly-Gly-Gly (YLGGG, 0.2 µM), whereas the corresponding sequence isomers Lys-Phe-Gly-Gly-Tyr (KFGGY, 0.3 nM) and Leu-Tyr-Gly-Gly-Gly (LYGGG, 1.2 nM) bound to Q8 with 1000-fold and 170-fold increases in affinity, respectively. To our knowledge, these are the highest affinities reported between a synthetic receptor and an unmodified peptide. The high-resolution crystal structures of the Q8·Tyr-Leu-Gly-Gly-Gly and Q8·Leu-Tyr-Gly-Gly-Gly complexes have enabled a detailed analysis of the structural determinants for molecular recognition. The high affinity, sequence-selectivity, minimal size of the target binding site, reversibility in the presence of a competitive guest, compatibility with aqueous media, and low toxicity of Q8 should aid in the development of applications involving low concentrations of target polypeptides.

Cucurbit[8]uril Binds Nonterminal Dipeptide Sites with High Affinity and Induces a Type II ß-Turn.

In an effort to target polypeptides at nonterminal sites, we screened the binding of the synthetic receptor cucurbit[8]uril (Q8) to a small library of tetrapeptides, each containing a nonterminal dipeptide binding site. The resulting leads were characterized in detail using a combination of isothermal titration calorimetry, 1H NMR spectroscopy, electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS), and X-ray crystallography. The equilibrium dissociation constant values determined for the binding of Q8 to nonterminal dipeptide sites Lys-Phe (KF) and Phe-Lys (FK) were 60 and 86 nm, respectively. These are to the best of our knowledge the highest affinities reported to date for any synthetic receptor targeting a nonterminal site on an unmodified peptide. A 0.79 Å resolution crystal structure was obtained for the complex of Q8 with the peptide Gly-Gly-Leu-Tyr-Gly-Gly-Gly (GGLYGGG) and reveals structural details of the pair-inclusion motif. The molecular basis for recognition is established to be the inclusion of the side chains of Leu and Tyr residues, as well as an extensive network of hydrogen bonds between the peptide backbone, the carbonyl oxygens of Q8, and proximal water molecules. In addition, the crystal structure reveals that Q8 induces a type II ß-turn. The sequence-selectivity, high affinity, reversibility, and detailed structural characterization of this system should facilitate the development of applications involving ligand-induced polypeptide folding.