Date of Award


Document Type

Thesis campus only



First Advisor

Adam R. Urbach


Chemists have long attempted to prepare synthetic receptors (SRs) that can bind natural targets with the high affinities and fidelities that antibodies bind antigens. This work describes the molecular recognition of methionine-terminated peptides by the SR cucurbit[8]uril (Q8) in aqueous solution with sub-micromolar affinity. Prior work established that Q8 binds with high affinity to peptides containing N-terminal aromatic amino acids, either by the simultaneous binding of two aromatic residues, one from each of two different peptides, or by the simultaneous binding of an aromatic residue and its immediate neighbor on the same peptide. The additional binding interface of two neighboring residues suggested the possibility of targeting nonaromatic peptides, which have thus far bound only weakly to SRs. A peptide library designed to test this hypothesis was synthesized and screened qualitatively for Q8 binding using a fluorescent indicator displacement assay. Additionally, the same peptide library was screened for binding to the acyclic cucurbit[n]uril-type receptors Zhang1 and Motor2, which have gained recent interest in solubilizing pharmaceutical drugs, but have not been fundamentally studied in peptide recognition. Although multiple peptides were found to potentially bind Zhang1 and Motor2, the screen results for Q8 prompted immediate follow up.

The large fluorescence response observed for several Met-terminated peptides suggested strong binding to Q8 and was confirmed quantitatively using isothermal titration calorimetry (ITC). The discovery of high affinity binding to Met-terminated peptides and, more generally, to non-aromatic peptides prompted a detailed investigation of the determinants of binding in this system using ITC, ESI-TOF-MS, and 1H NMR spectroscopy for 25 purified peptides. The studies establish the sequence determinants required for high-affinity binding of Met-terminated peptides and demonstrate that cucurbit[n]uril-mediated peptide recognition does not require an aromatic residue for high affinity. These results, combined with the known ability of cucurbit[n]urils to target N-termini and disordered loops in folded proteins, suggest that Q8 could be used to target Met-terminated proteins. In order to test this hypothesis, we are now engineering minimal affinity tags on human carbonic anhydrase-II (HCA). In our cloning and expression strategy, HCA is designed to contain an N-terminal purification tag followed by an intein sequence, which cleaves itself after purification to leave an exposed, N-terminal Q8 binding site. This work describes the successful cloning of the HCA gene into the inteincontaining plasmid. Expression of the recombinant HCA proteins is now underway and binding studies with Q8 will be pursued.