Correlation of Ice Recrystallization Inhibition Activity to Hydration Parameters and Effect of the C-3 Hydroxyl Group

Abstract

The use of cryoprotective agents (CPAs) in the storage of cells and tissues is essential for transfusion medicine and food storage. Currently, cryoprotectants such as glycerol and DMSO are ineffective at preventing ice recrystallization during freezing and thawing. Ice recrystallization is a significant contributor to cellular injury because ice growth can cause mechanical damage to cells. Modified CPAs that inhibit ice recrystallization are advantageous for improving cell recovery and viability following cryopreservation. Over the years, the Ben Lab has developed ice recrystallization inhibitors (IRIs) inspired by natural biological antifreezes. Further research has led to the discovery of small carbohydrate-based molecules that can be used as active IRIs. The Ben Lab has previously shown that hydration parameters can be used to estimate IRI activity. Hydration parameters such as hydration number, hydration index, and partial molar compressibility have been found to be positively correlated to IRI activity for simple carbohydrates. However, these parameters cannot accurately account for the hydrophobic nature of functionalized carbohydrate derivatives. Therefore, a predictive hydration parameter for IRI activity must be able to describe carbohydrates that have both hydrophobic and hydrophilic regions. It is hypothesized that the partition coefficient could be used to assess the activity of functionalized carbohydrates because it provides a quantitative measure of a compound’s hydrophilicity and hydrophobicity. The first part of this thesis will look at several hydration parameters and their correlation to half-maximal inhibitory concentration (IC50) values obtained from the 5-minute modified “splat-cooling” assay. These parameters have previously correlated to percent mean iv grain size (%MGS) values (obtained from the 30-minute modified “splat-cooling” assay) and will act as a point of comparison for the IC50 activity data. In addition to the previously tested parameters, the log of the partition coefficient (logP) relationship with IRI activity will be explored. The correlations between hydration parameters and IRI activity measured using IC50 values are not the same as those measured with %MGS values. The logP hydration parameter displays a negative correlation with IRI activity measured using IC50 values in which high IRI activity is associated with low logP values. The second part of this thesis will examine the impact of functionalization at the C-3 hydroxyl position of glucosides on IRI activity. The C-1 and C-6 hydroxyl positions have been extensively analyzed for their effects on IRI activity, but the C-3 position has been left primarily uninvestigated. C-3 functionalized compounds are synthesized and compared to their corresponding C-1 functionalized compounds. The results from the study illustrate the C-3 functionalized compounds have poor solubility compared to the C-1 compounds, which emphasizes the importance of the C-2 and C-4 positions in the hydration of carbohydrates. The range of IRI activity for the C-3 position is of interest because this position is more sensitive to functionalization than the C-1 position. Collectively, these results illustrate that logP can be used as a potential predictor of IRI activity and the importance of the C-2 and C-4 positions for hydration and solubility following investigation into modifications of the C-3 position. The C-3 position has a unique characteristic of being receptive to functionalization and is essential for the rational future design of IRIs.