C-linked AFGP analogues containing beta-amino acids: Preparation, assessment and in vitro studies

Abstract

Antifreeze Glycoproteins (AFGPs) are peptide-based compounds present in the blood serum of arctic and antarctic fish. These compounds inhibit the growth of ice and are key to their survival in icy cold water. The mechanism by which they inhibit the ice growth is regarded as an adsorption-inhibition process at the macromolecular level. However, the mechanism of action at the molecular level is still under debate among researchers. Our laboratory has been designing C-Linked AFGP analogues possessing enhanced chemical and biological stability for the purpose of structure-activity relationship studies. Towards this end, a series of AFGP analogues containing a beta-amino acid in the polypeptide backbone have been synthesized. The requisite glycosylated beta-amino acids in the building blocks were synthesized via a modified Arndt-Eistert homologation. These building blocks were then assembled into antifreeze analogues using standard solid phase synthesis protocols. These analogues were evaluated for antifreeze-specitic activity that is, for recrystallization inhibition (RI) and thermal hysterisis (TH). The analogues demonstrated a significant degree of TH, dynamic ice shaping and genuine RI activity attributed to biological antifreezes. Solution conformation of new analogues was performed using circular dichroism (CD) spectroscopy. Results showed that the overall conformation is mainly random coil. In vitro cytotoxicity studies of these analogues at both physiological and cryogenic temperatures were performed on a human embryonic liver cell line. Except analogue 74, the other analogues were found to be cytotoxic and cryotoxic at high analogue concentrations.