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.

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 (Kd) values. Unexpectedly, the Kd 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 Kd 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.