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

Abstract

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 Phe^B1 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.

Figure 1.

(Top left) Chemical formula of Q7; (bottom left) schematic of the binding of N-terminal Amf to Q7, showing inclusion of the aromatic side chain and interaction of the α-ammonium and side-chain ammonium groups with opposite portals of Q7 (shown in red); (right) model of the Q7·AM-insulin complex based on the crystal structure of the Q7·insulin complex (PDB ID3Q6E).

Figure 2.

Four-step semisynthesis of AM-insulin from native insulin. Abbreviations: Msc-ONp, methyl sulfonylethyl p-nitrophenyl carbonate; Et3N, triethylamine; r.t., room temperature; PITC, phenyl isothiocyanate; TFA, trifluoroacetic acid; NEM, N-ethyl morpholine.

Figure 3.

Overlay of UV circular dichroism spectra of insulin (open ovals) and AM-insulin (filled ovals) at 25 °C in 10 mM sodium phosphate, 1 mM lysine, 4 mM EDTA, pH 7.0.

Figure 4.

Deconvoluted ESI-TOF mass spectra of aqueous samples of (a) AM-insulin, calc m/z 5837.76, and (b) an equimolar mixture of AM-insulin and Q7, calc m/z 7000.10.

Figure 5.

Representative ITC data of the titration of AM-insulin with Q7 at 300 K in 10 mM sodium phosphate, pH 7.0.