Site specific introduction of unnatural amino acids at sites critical to insulin receptor recognition & biological activity

Insulin constitutes a hormone of central importance in physiology and a vital element in glucose management. Its use in diabetes care has been of seminal significance for nearly a century. The advent of chemical biotechnology (biosynthesis with unnatural amino acids) provides a new approach for optimizing insulin pharmacology through the use of previously unexplored chemical diversity.;This thesis focuses on the C-terminal region of the insulin B-chain and in particular two aromatic amino acids central to insulin bioactivity, B 24 and B25. The selective introduction of a series of Phe-related derivatives at each position has yielded an insightful perspective on insulin receptor recognition and the basis for selectivity relative to the homologous IGF-I receptor. Position 24 has proven highly restrictive to structural change and the results emphasize the importance of backbone conformation to full potency. In comparison, position B25 is quite accommodating to sizable changes in side-chain structure as long as they maintain an aromatic character, but highly demanding in the specific distance of the phenyl ring from the peptide backbone. These collective observations establish a foundation for application of unnatural amino acids as a route to insulin pharmacology that may not be obtainable with natural amino acids.;A set of AspB10 and Gly24-based insulin analogs were studied as a means to assess the interaction of two linearly distant amino acids that synergistically influence insulin bioactivity. While position B24 proved highly restrictive to structural change the combination with AspB10 yielded a variable increase in bioactivity in the range of 2-50 fold dependent on the specific B24 amino acid. The improved activity was a direct function of AspB10 and could not be translated to provide similar influence to B24 analogs that were of non-aromatic character or of initial high potency. Interestingly, the specific placement of a single methyl group at different positions on the B24 phenylalanine ring generated sizable differences in activity. The molecular basis of this synergism remains undefined.;A specific lactam-brdige was introduced to the C-terminal region surrounding the two aromatic amino acids of central importance to bioactivity. The synthesis proved challenging and only achieved through the application of native single chain insulin synthesis. The single chain lactam-brdige insulin was observed to be poorly active based on the binding affinity assay and was not improved by the enzymatic conversion to the native insulin two chain structure. These observations emphasize the importance of maintaining the native backbone structure in this area with conformational restriction serving to significantly diminish bioactivity.