A major problem associated with cellular cryopreservation is the recovery of cellular viability upon thawing. Current cryopreservation techniques use additives such as DMSO, sucrose and fetal bovine serum. However, each have their own respective cytotoxic issues. A significant factor in cryotoxicity is the formation of large ice crystals which can damage cellular components and cause dehydration. This has significant impacts for applications such as food preservation, scientific research, and tissue preservation.
To this end, our laboratory is interested in synthesizing biologically-relevant compounds that can act as cryoprotectants by preventing the formation of large ice crystals in sub-zero temperatures. Our lab has previously synthesized structural analogues of native antifreeze glycoproteins (AFGPs, found in the blood of Antarctic cod), that possess the unique ability to inhibit ice-recrystallization. However, the mechanism by which they inhibit ice recrystallization is unclear.
This thesis focuses on efforts made to understand this mechanism, and synthesize molecules that are more potent in ice recrystallization inhibition (IRI) activity compared to previously synthesized analogues.
By assessing the IRI activity of various mono- and disaccharides, we have shown that the density of water molecules that surround a carbohydrate (Hydration Index) is directly proportional to the ability of sugars to inhibit ice crystal growth.
In an effort to design functional AFGP analogues, various C-linked analogues were synthesized that contained different spacer lengths between the carbohydrate and the peptide backbone. Analyses of the solution conformations of these analogues showed that IRI-active AFGP analogues contain a distinct conformation in which the carbohydrate is oriented to form a hydrophobic pocket with the side chain. We hypothesize that this change in glycoconjugate hydration is responsible for disturbing its surrounding waters, thereby preventing water from adding to the ice lattice required for ice growth.
Finally, SAR studies showed that threonine-containing AFGP and antifreeze proteins are more potent in antifreeze activity than serine-containing analogues. As the most potent AFGP analogue previously synthesized by our lab contains a C-linked-α-galactosyl -serine residue, we hypothesized that the analogous glycopeptide containing a C-α-galactosyl-threonine residue will be more potent in antifreeze activity. The final section describes efforts to synthesize a C-linked α-galactosyl-threonine glycoconjugate.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OOU.#10393/19739 |
| Date | 04 February 2011 |
| Creators | Tam, Roger Y. |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | thesis |
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