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In this study, N -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 N -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 N -methylated peptides. Additionally, circular dichroism (CD) measurements were used to confirm that aggregation processes did not occur in the presence of the N -methylated analogs of hot-spot insulin fragments, and that insulin retains its native conformation. Of the seven N -methylated analogs of the A- and B-chain hot-spots of insulin, six inhibited insulin aggregation (peptides 1 and 3 - 7 ). 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., Competing Interests: The authors declare no conflict of interest.

This study investigates the propensity of short peptides to self-organize and the influence of aggregates on cell cultures. The dipeptides were derived from both enantiomers of identical aromatic amino acids and tripeptides were prepared from two identical aromatic amino acids with one cysteine or methionine residue in the C-terminal, N-terminal, or central position. The formation or absence of fibrous structures under physiological conditions was established using Congo Red and Thioflavine T assays as well as by microscopic examination using normal and polarized light. The in vitro stability of the aggregates in buffered saline solution was assessed over 30 days. Materials with potential for use in regenerative medicine were selected based on the cytotoxicity of the peptides to the endothelial cell line EA.hy 926 and the wettability of the surfaces of the films, as well as using scanning electron microscopy. The criteria were fulfilled by H- d Phe d Phe-OH, H- d Cys d Phe d Phe-OH, H-CysTyrTyr-OH, H- d Phe d Phe d Cys-OH, H-TyrTyrMet-OH, and H-TyrMetTyr-OH. Our preliminary results suggest that the morphology and cell viability of L919 fibroblast cells do not depend on the stereochemistry of the self-organizing peptides.

We have shown for the first time that a natural protein (human insulin) can be acylated at the N‐terminus with a β‐amino acid (H‐β3hAla‐), in a process catalyzed by the β‐peptidyl aminopeptidase 3‐2W4‐BapA. This selective modification, which could also be applied for protein labeling and tagging, should be generally useful, also to protect peptides and proteins from attack by common aminopeptidases. [ABSTRACT FROM AUTHOR]