Sequence-Specific Inhibition of a Nonspecific Protease

A nonspecific exopeptidase, aminopeptidase N (APN), is inhibited sequence-specifically by a synthetic host, cucurbit[7]­uril (Q7), which binds with high affinity and specificity to N-terminal phenylalanine (Phe) and 4-(aminomethyl)­phenylalanine (AMPhe) and prevents their removal from the peptide. Liquid chromatography experiments demonstrated that in the presence of excess Q7, APN quantitatively converts the pentapeptides Thr-Gly-Ala-X-Met into the dipeptides X-Met (X = Phe, AMPhe). The resulting Q7-bound products are completely stable to proteolytic digestion for at least 24 h. Structure–activity studies revealed a direct correlation between the extent of protection of an N-terminal amino acid and its affinity for Q7. Therefore, Q7 provides predictable sequence-specificity to an otherwise nonspecific protease and enables the production of a single peptide product. Conversely, APN uncovers a high-affinity epitope that is subsequently bound by Q7, and thus this approach should also facilitate the molecular recognition of peptides

Nanomolar Binding of Peptides Containing Noncanonical Amino Acids by a Synthetic Receptor

This paper describes the molecular recognition of phenylalanine derivatives and their peptides by the synthetic receptor cucurbit[7]uril (Q7). The 4-<i>tert</i>-butyl and 4-aminomethyl derivatives of phenylalanine (tBuPhe and AMPhe) were identified from a screen to have 20–30-fold higher affinity than phenylalanine for Q7. Placement of these residues at the N-terminus of model tripeptides (X-Gly-Gly), resulted in no change in affinity for tBuPhe-Gly-Gly, but a remarkable 500-fold increase in affinity for AMPhe-Gly-Gly, which bound to Q7 with an equilibrium dissociation constant (<i>K</i>_d) value of 0.95 nM in neutral phosphate buffer. Structure–activity studies revealed that three functional groups work in a positively cooperative manner to achieve this extraordinary stability (1) the N-terminal ammonium group, (2) the side chain ammonium group, and (3) the peptide backbone. Addition of the aminomethyl group to Phe substantially improved the selectivity for peptide versus amino acid and for an N-terminal vs nonterminal position. Importantly, Q7 binds to N-terminal AMPhe several orders of magnitude more tightly than any of the canonical amino acid residues. The high affinity, single-site selectivity, and small modification in this system make it attractive for the development of minimal affinity tags

Cucurbit[7]uril–Tetramethylrhodamine Conjugate for Direct Sensing and Cellular Imaging

This paper describes the design and synthesis of a conjugate (Q7R) comprising the synthetic host cucurbit[7]­uril (Q7) linked to the fluorescent dye tetramethylrhodamine (TMR), and the characterization of its optical and guest-binding properties as well as its cellular uptake. Q7R was synthesized in two steps from monofunctionalized azidobutyl-Q7 and NHS-activated TMR. The fluorescence of Q7R is quenched upon guest binding, and this observable was used to determine equilibrium dissociation constant (<i>K</i>_d) values. Unexpectedly, the <i>K</i>_d values for guests binding to Q7R and to unmodified Q7 were essentially identical. Therefore, Q7R can directly report binding to Q7 without an energetic penalty due to the conjugated fluorophore. This result demonstrates a potentially general strategy for the design of single-component host–indicator conjugates that respond sensitively to analytes without perturbing the binding properties of the host. The unique properties of Q7R enabled measurement of <i>K</i>_d values across 3 orders of magnitude and at concentrations as low as 0.7 nM. This result is particularly relevant given the unmatched range of guests and binding affinities demonstrated for Q7. Confocal fluorescence microscopy of live and fixed HT22 neurons revealed the cellular uptake of Q7R and its punctate localization in the cytoplasm. Q7R did not alter cell growth at concentrations up to 2.2 μM over 4 days. These experiments demonstrate the feasibility of Q7R as a direct sensor for guest binding and as a cell-permeable compound for imaging applications