Improving the rational design of antifreeze glycoproteins through identification of the parameters that influence ice recrystallization inhibition

Antifreeze glycoproteins (AFGPs) are a subclass of biological antifreezes isolated from Antarctic and Atlantic Teleost fish. These compounds have the ability to depress the freezing points of solutions as well inhibit ice crystal growth, thereby protecting fish from cryoinjury and death. Although native AFGPs have considerable promise as cryoprotectants, their limited bioavailability, cytotoxicity, biological instability and lengthy chemical syntheses have precluded their Widespread use. Consequently, Ben et al. have designed non-cytotoxic stable C-linked AFGPs with comparable ice recrystalization inhibition (RI) to that of the natural compounds. This work also revealed that amongst glycopeptides with the same amino acid backbone, the overall recrystalization inhibition activity correlates to the hydration of the carbohydrate moiety. This interesting relationship lead to question if whether the hydration values of carbohydrates could be used to predict the activity of synthetic antifreeze glycoproteins. If true, it would greatly improve the rational design of synthetic analogues. Therefore the relationship between hydration and ice recrystalization inhibition was studied using small molecule carbohydrates. These results were also compared to the current standard in cryoprotection of tissues and cells, dimethyl sulfoxide, and C-linked carbohydrate derivatives. The study elucidates that there is a more accurate parameter then hydration to predict the ice recrystalization inhibition of carbohydrates, and it was defined as the Hydration Index .;Another key component in improving the rational design of syAFGPs as cryoprotectants is to distinguish between the structural motifs necessary for ice recrystalization inhibition and thermal hysteresis. In 2004, Nishimura et al. published such work on the essential components of AFGPs for thermal hysteresis. They reported that even small changes to the native AFGPs can cause a loss of thermal hysteresis, for example exchanging the naturally occurring threonine residues for serine. Unfortunately, they did not test any of their compounds for recrystallization inhibition. Opportunely, the synthesis towards an antifreeze glycoprotein which contains serine residues in place of threonine residues is presented, with the future goal of testing for its RI activity.;The overall objective of this dissertation is to probe the characteristics that dictate antifreeze glycoprotein activity in terms of ice recrystalization inhibition, with the goal of providing useful information towards the rational design of potent non-toxic cryoprotectants.;Furthermore, the investigation of carbohydrates with respect to RI was extended to the disaccharide present in the native antifreeze glycoproteins, D-galactose-beta(1-3)-N-acetyl-galactosamine. Its vital importance in AFGPs is apparent as no synthetic AFGP (syAFGP) with a carbohydrate other then that found in the native disaccharide has been able to surpass the activity of the natural compounds. Yet the intrinsic properties of the disaccharide, separated from the peptide, have never been studied in terms of RI activity. Therefore, the synthesis of the native disaccharide and analysis of its RI activity would help to determine how much of a factor it is for the overall activity of the natural AFGP. This work also creates the opportunity to study how other aspects of D-galactose-beta(1-3)-N-acetyl-galactosamine effects ice recrystalization inhibition, such as the C2 N-acetyl moiety and the regiochemistry of the glycosidic linkage.