Examining the role of carbohydrate hydration and structure in preventing ice-recrystallization

• Main Author: Chaytor, Jennifer L
• Format: Thesis
• Language: English
• Published: University of Ottawa (Canada) 2010
• Subjects: Chemistry; Organic; Antarctic; Antarc*
• Online Access: http://hdl.handle.net/10393/29967; https://doi.org/10.20381/ruor-20005

Antifreeze glycoproteins (AFGPs) are a subclass of biological antifreezes that are found in many species of Antarctic and Atlantic teleost fish. These compounds restrict the growth of ice and therefore protect these organisms from cryo-injury and death. Our laboratory is exploring the rational design of chemically and biologically stable AFGPs that have biological activity and therefore have potential medical and industrial applications. To this end, we have prepared a series of C-linked AFGP analogues that possess thermal hysteresis (TH) and ice-recrystallization inhibition (IRI) activity. This thesis examines the effect of stereochemical and structural modifications of carbohydrates on IRI activity. Recently, our laboratory evaluated several commercially available O-linked mono- and disaccharides with varying stereochemical configurations for antifreeze activity. It was discovered that these sugars do not display thermal hysteresis activity, but do display IRI activity. Furthermore, their IRI activity is related to their solvation or hydration, which affects their ability to fit into the three-dimensional hydrogen-bonded network of ice. The ability of a sugar to be solvated depends on the stereochemical relationship of the hydroxyl groups on C2 and C4. The IRI activity of these mono- and disaccharides was then correlated to their ability to protect human embryonic kidney cells at sub-zero temperatures. It has been proposed that the majority of damage to cells and tissues is caused by the recrystallization of ice during freeze-thaw protocols. Carbohydrates have been used as cryoprotective agents in the past, however, a systematic study that compares the IRI activity of these compounds to their cryoprotective ability has not been reported. Our observations support the theory that ice-recrystallization is a major cause of damage to cells during the freeze-thaw cycle, and that we can design novel cryoprotectants based upon the results of our IRI activity assessment. I have recently prepared several new classes of substituted C-linked carbohydrates, which have been designed to disrupt the hydration of carbohydrates through hydrophobic interactions. It is believed that hydrophobic groups will affect the fit of these compounds into the three-dimensional hydrogen-bonded network of water, and therefore change their ability to inhibit the recrystallization of ice. In the first series, the IRI activity of compounds with an alpha C-allyl group mirrored the trend of their O-linked parent monosaccharides. Through the synthesis of these and other compounds, it was found that for galactose, the anomeric stereochemistry does playa role in RI activity. Methyl-alpha/beta-galactopyranoside has been observed to have a poor compatibility with the three-dimensional hydrogen-bonded structure of ice. Compounds with a methoxy group at C2, C3, C4, and C6 respectively were prepared using standard protecting group chemistry. These compounds form the basis of a second series of C-linked carbohydrates that all showed a decrease in IRI activity compared to a non-methylated control. These results suggest that the methyl group disturbs the hydration of the carbohydrate and affects its ability to interact with ice. Interestingly, when longer groups (ethyl, propyl, butyl, and hexyl) were added to C2 to form a third series of compounds, we saw a steady increase in IRI activity. These compounds may be forming liquid-crystals or gelators, which is affecting their hydration and increasing their interaction with the ice-water interface. Thioglycoside derivatives of galactose were also found to have moderate IRI activity. In another drastic structural variation, we examined the addition of a geminal-difluoromethylene group at the anomeric position in a fourth series of C-linked compounds. The results of the IRI assay of fluorinated sugars suggest that the fluorine atoms do not result in increased IRI activity. A fluorinated sugar showed lower levels of IRI activity when compared to an appropriate non-fluorinated control. In order to develop an understanding of the role that biological antifreezes play at the ice-water interface, we undertook a series of Coherent Anti-Stokes Raman Scattering (CARS) experiments to observe the OH-stretching regions of solutions of water and biological antifreezes at sub-zero and room temperatures. The results of these experiments will be described in this dissertation.