Microwave assisted SPPS of amylin and its toxicity of the pure product to RIN-5F cells.

摘要

The 37-amino acid polypeptide islet amyloid polypeptide (IAPP), or amylin, is found as amyloid aggregates in the islets of Langerhans in patients with type II diabetes. Herein, we report an efficient microwave assisted solid phase peptide synthesis of amylin (IAPP). The most efficient synthesis used double and triple couplings and 10 equiv. of amino acids. Double couplings were used for most amino acids, whereas triple couplings were utilized for amino acids in selected regions. The most effective method for formation of the disulfide bond in amylin was found to be iodine oxidation. The highest purity amylin was obtained when the crude peptide was purified with HPLC before formation of the disulfide bond. The cytotoxicity of the synthesized amylin product to RIN-5F cells was determined. The synthesized amylin exhibits an exponential increase of cytotoxicity at concentrations >35 microM. Transmission electron microscope studies of a sample of amylin shows that insoluble amyloid fibrils spontaneously formed when 45 microM solution of synthesized amylin was incubated in a suitable buffer for 6 h.