Developing Novel Peptide Ligation Methodology for the Synthesis and Labelling of Bioactive Hepcidin

Hepcidin is a 25 amino-acid peptide hormone with an essential role in the systemic regulation of iron. The dysregulation of hepcidin is linked to a number of iron loading disorders such as hereditary haemochromatosis and anaemia of inflammation. Procuring synthetic, recombinant or endogenous hepcidin is challenging and expensive; therefore strategies to improve the synthesis of hepcidin and add modifications to extend its short half-life in vivo are required for potential therapeutic applications. \nIndustrial scale production of a peptide such as hepcidin for therapeutic use would likely be performed using a convergent approach, involving the ligation of peptide fragments. Native chemical ligation (NCL) is a powerful tool in peptide and protein synthesis, allowing peptide fragments to be joined together through a chemoselective ligation between a peptide-thioester and a cysteinyl peptide. Preparation of peptide¬thioesters is challenging, however thioester formation via 1 U 6 acyl transfer at cysteine residues allows generation of thioesters in both synthetic and recombinant peptides and proteins. \nHere, investigations into optimising the preparation of peptide-thioesters via 1 U 6 acyl transfer have been carried out to extend its application to the synthesis of complete hepcidi n, analogues and labelled variants. This led to the development of a novel, faster route to peptide thioesters via 1 U 6e acyl transfer, which was applied to the cyclisation of peptides via in situ NCL following thioester formation. \nAdditionally a novel methodology via 1 U 6 acyl transfer has been established for the preparation of peptide hydrazides, which are stable alternatives to peptide-thioesters in NCL. These novel strategies have been applied to full-length hepcidin and fragments of hepcidin, which in conjunction with NCL, have been used to generate a variety of hepcidins. A measurable reduction in the iron efflux membrane protein, ferroportin, in cells treated with these analogues, confirmed their biological activity, indicating hepcidin-induced internalisation of ferroportin, including for hepcidin biotinylated using the hydrazide-NCL strategy. \n