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.

Semisynthesis of Aminomethyl-Insulin: An Atom-Economic Strategy to Increase the Affinity and Selectivity of a Protein for Recognition by a Synthetic Receptor.

Advancements in the molecular recognition of insulin by nonantibody-based means would facilitate the development of methodology for the continuous detection of insulin for the management of diabetes mellitus. Herein, we report a novel insulin derivative that binds to the synthetic receptor cucurbit[7]uril (Q7) at a single site and with high nanomolar affinity. The insulin derivative was prepared by a four-step protein semisynthetic method to present a 4-aminomethyl group on the side chain of the PheB1 position. The resulting aminomethyl insulin binds to Q7 with an equilibrium dissociation constant value of 99 nM in neutral phosphate buffer, as determined by isothermal titration calorimetry. This 6.8-fold enhancement in affinity versus native insulin was gained by an atom-economical modification (-CH2NH2). To the best of our knowledge, this is the highest reported binding affinity for an insulin derivative by a synthetic receptor. This strategy for engineering protein affinity tags induces minimal change to the protein structure while increasing affinity and selectivity for a synthetic receptor.

Revisiting Fluorescent Calixarenes: From Molecular Sensors to Smart Materials.

Calix[n]arenes (n = 4, 5, 6, 8) are "chalicelike" phenol-based macrocycles that are among the most fascinating and highly studied scaffolds in supramolecular chemistry. This stems from the functional and tunable diversity at both their upper and lower rims, their preorganized nonpolar cavities and preorganized ion-binding sites, and their well-defined conformations. Conjugation of calixarene scaffolds with various fluorogenic groups has led to the development of smart fluorescent probes that have been utilized as molecular sensors, in bioimaging, for drug and gene delivery, in self-assembly/aggregation, and as smart materials. The fine-tuning and incorporation of different ligating sites in the calix[4]arene scaffold have produced numerous molecular sensors for cations, anions, and biomolecules. Moreover, the aqueous solubility of p-sulfonatocalix[4]arenes has engendered their potential use in drug/gene delivery and enzymatic assays. In addition, because of their strong optical properties, fluorescent calix[4]arenes have been used to develop smart materials, including gels as well as nonlinear optical, organic light-emitting diode, and multiphoton materials. Finally, significant developments in the utility of fluorescent higher calixarenes have been made for bioapplications. This review critically summarizes the recent advances made in all of these different areas.

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.

On the Nature of Guest Complexation in Water: Triggered Wetting-Water-Mediated Binding.

The complexity of macromolecular surfaces means that there are still many open questions regarding how specific areas are solvated and how this might affect the complexation of guests. Contributing to the identification and classification of the different possible mechanisms of complexation events in aqueous solution, and as part of the recent SAMPL8 exercise, we report here on the synthesis and conformational properties of TEEtOA 2, a cavitand with conformationally flexible ethyl groups at its portal. Using a combination of ITC and NMR spectroscopy, we report the binding affinities of a series of carboxylates to 2 and compare it to a related cavitand TEMOA 1. Additionally, we report MD simulations revealing how the wetting of the pocket of 2 is controlled by the conformation of its rim ethyl groups and, correspondingly, a novel triggered wetting, guest complexation mechanism, whereby the approaching guest opens up the pocket of the host, inducing its wetting and ultimately allows the formation of a hydrated host-guest complex (H·G·H2O). A general classification of complexation mechanisms is also suggested.

Cavitand Complexes in Aqueous Solution: Collaborative Experimental and Computational Studies of the Wetting, Assembly, and Function of Nanoscopic Bowls in Water.

Water is the dominant liquid on Earth. Despite this, the main focus of supramolecular chemistry research has been on binding and assembly events in organic solvents. This arose because it is more straightforward to synthesize organic-media-soluble hosts and because of the relative simplicity of organic solvents compared to water. Nature, however, relies on water as a solvent, and spurred by this fact, supramolecular chemists have recently been making forays into the aqueous domain to understand water-mediated non-covalent interactions. These studies can benefit from the substantial understanding of the hydrophobic effect and electrostatic interactions developed by physical chemists. Nearly 20 years ago, the Gibb group first synthesized a class of water-soluble host molecules, the deep-cavity cavitands, that possess non-polar pockets that readily bind non-polar moieties in aqueous solution and are capable of assembling into a wide range of complexes with distinct stoichiometries. As such, these amphipathic host species are ideal platforms for studying the role of negatively curved features on guest complexation and the structural requirements for guided assembly processes driven by the hydrophobic effect. Here we review the collaborative experimental and computational investigations between Gibb and Ashbaugh over the past 10 years exploring questions including the following: How does water wet/solvate the non-polar surfaces of non-polar pockets? How does this wetting control the binding of non-polar guests? How does wetting affect the binding of anionic species? How does the nature and size of a guest size impact the assembly of cavitand hosts into multimeric capsular complexes? What are the conformational motifs of guests packed within the confines of capsular complexes? How might the electrostatic environment engendered by hosts impact the properties and reactivity of internalized guests?

Proximal charge effects on guest binding to a non-polar pocket.

Science still does not have the ability to accurately predict the affinity that ligands have for proteins. In an attempt to address this, the Statistical Assessment of Modeling of Proteins and Ligands (SAMPL) series of blind predictive challenges is a community-wide exercise aimed at advancing computational techniques as standard predictive tools in rational drug design. In each cycle, a range of biologically relevant systems of different levels of complexity are selected to test the latest modeling methods. As part of this on-going exercise, and as a step towards understanding the important factors in context dependent guest binding, we challenged the computational community to determine the affinity of a series of negatively and positively charged guests to two constitutionally isomeric cavitand hosts: octa-acid 1, and exo-octa acid 2. Our affinity determinations, combined with molecular dynamics simulations, reveal asymmetries in affinities between host-guest pairs that cannot alone be explained by simple coulombic interactions, but also point to the importance of host-water interactions. Our work reveals the key facets of molecular recognition in water, emphasizes where improvements need to be made in modelling, and shed light on the complex problem of ligand-protein binding in the aqueous realm.