This presentation will describe the sequence-specific, nanomolar binding of tripeptides by the synthetic receptor cucurbit[8]uril (Q8), which is large enough to accommodate two guests within its cavity. This uniquely strong and specific recognition is made possible by the folding and inclusion of two neighboring amino acid side chains within the Q8 cavity. The fluorescent dye tetramethylbenzobis(imidazolium) (MBBI) was used as a sensor component to study the binding of the Q8:MBBI complex to a library of 105 peptides that vary in the N-terminal residue (tyrosine, phenylalanine, and tryptophan) and in the identity of the neighboring residues (varied to 18 amino acids at each position). The fluorescence intensity of Q8:MBBI increased upon treatment with the peptide Tyr-Leu-Ala (YLA), indicating displacement of MBBI from the Q8 cavity, but decreased upon treatment with its sequence isomer Tyr-Ala-Leu (YAL), indicating binding of the tripeptide inside the Q8:MBBI complex. Further studies by <sup>1</sup>H NMR spectroscopy, mass spectroscopy, isothermal titration calorimetry, and fluorescence spectroscopy show that Tyr-Leu-Ala binds to Q8 in a 1:1 stoichiometric ratio, with an unprecedented dissociation constant of 7.2 nM, and with &gt;4000-fold specificity versus Tyr-Ala-Leu due to the inclusion of the side chains of the neighboring Tyr and Leu residues in Tyr-Leu-Ala. This novel binding mode was confirmed through the use of Rotating Frame Nuclear Overhauser-Effect Spectroscopy (ROESY) to identify protons that were located proximally though space for the Q8:YLA complex.

Sequence-specific, nanomolar peptide recognition via the folding and inclusion of neighboring sidechains in cucurbit[8]uril

249th National Meeting (2015)

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