Insulin Hot-Spot Analogs Formed with N-Methylated Amino Acid Residues Inhibit Aggregation of Native Hormone

Abstract

In this study, <i>N</i>-methylated analogs of hot-spots of insulin were designed and synthesized, in the expectation that they would inhibit the aggregation of both insulin hot-spots and the entire hormone. Synthesis of insulin "amyloidogenic" analogs containing <i>N</i>-methylated amino acid residues was performed by microwave-assisted solid phase according to the Fmoc/tert-Bu strategy. As a coupling reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TosO^-) was used. Three independent methods were applied in aggregation studies of the complexes of insulin with its <i>N</i>-methylated peptides. Additionally, circular dichroism (CD) measurements were used to confirm that aggregation processes did not occur in the presence of the <i>N</i>-methylated analogs of hot-spot insulin fragments, and that insulin retains its native conformation. Of the seven <i>N</i>-methylated analogs of the A- and B-chain hot-spots of insulin, six inhibited insulin aggregation (peptides <b>1</b> and <b>3</b>-<b>7</b>). All tested peptides were found to have a lower ability to inhibit the aggregation of insulin hot-spots compared to the capability to inhibit native hormone aggregation.