Synthesis of Nitrogen-Containing Carbohydrate Derivatives and Their Use Toward Inhibiting Ice Recrystallization and Gas Hydrate Formation

Ice recrystallization during cryopreservation results in cell death and decreased cell viabilities due to cellular damage. This is a significant problem particularly in regenerative medicine where decreased cell viabilities post-thaw affect the success of the therapy. Given the success of these therapies to treat various diseases, the development of novel cryprotectants which have the ability to inhibit ice recrystallization during freezing and thawing are urgently required. Current cryoprotectant such as dimethyl sulfoxide, is associated with cytotoxicity in the clinical settings and thus are not optimal cryoprotectants. Our laboratory is interested in the rational synthesis of non-cytotoxic small molecules which possess the property of ice recrystallization inhibition (IRI) activity. \ud Previously, the Ben laboratory has demonstrated that simple monosaccharides possess moderate ice recrystallization inhibition activity and that this activity is linked to hydration. The “compatibility” of the carbohydrate within the three-dimensional hydrogen bonded network of water is inversely proportional to its IRI activity. Hydration has previously been directly linked to the stereochemical relationship of individual hydroxyl groups on the carbohydrate. Additionally, it has been proposed that intramolecular hydrogen bond formation and hydrogen bonding cooperativity has a large effect on the water structure thus impacting hydration. Structure-function work has suggested that the presence of an amine as a hydrogen donor at the endocyclic position within the pyranose ring maybe beneficial to IRI activity. Thus, the first part of this thesis describes the synthesis and IRI activity of D-glucose and D-galactose based azasugars and its analogues. These azasugars have replaced the endocyclic ring oxygen with an amine. These azasugars and their analogues were found to possess moderate to potent IRI activity suggesting that hydrogen bond donation may be important for hydration and thus, IRI activity at the endocyclic ring oxygen. \ud During the development of these azasugars, the Ben laboratory developed carbohydrate-based surfactants and hydrogelators possessing unprecedented IRI activity. A potential use of molecules possessing IRI activity is towards the inhibition of gas hydrate formation. Gas hydrates are ice-like solids containing gases within a highly ordered network of water molecules. These gas hydrates tend to accumulate in oil and gas pipelines posing significant dangers as the build-up of solid material leads to blockages in the pipeline reducing flow. Previous work had demonstrated the use of antifreeze proteins possessing potent IRI activity in inhibiting gas hydrate formation. However, their complex structure limits commercial use. Thus, the second part of the thesis describes the use of the azasugars, carbohydrate-based surfactants and hydrogelators in inhibiting gas hydrate formation. The effectiveness of the small molecules is compared to a commercial inhibitor PVP 10. Some of these small molecules were significantly better inhibitors of gas hydrate formation than the currently utilized inhibitor PVP 10. The low molecular weights of these small molecules, easy synthesis and potency make them excellent alternatives to PVP 10. However it was found that while some of the structural features in the small molecules may be amenable to both activities, it seems that the ability to inhibit ice recrystallization is not a good indicator of a compounds ability to inhibit gas hydrate formation. \ud In a continuing effort to develop novel small molecule IRIs, the Ben laboratory has develop three classes of compounds. These include: carbohydrate-based surfactants and hydrogelators, lysine-based surfactants and truncated C-linked glycopeptides. Structure-function work utilizing these compounds revealed that presence of long alkyl chains, an amide linkage and the presence of an open-alditol chain are all important to IRI activity. However, the surfactant-like nature limits their use in cryopreservation and thus prompted the discovery of phenoxyglycosides as IRI active molecules. The structural features of these recently developed small molecules were combined to generate novel small molecule IRIs which do not resemble surfactants. These novel small molecules included “disaccharides” which possessed an aryl group at the anomeric position of a pyranose ring and an open-alditol chain linked via an amide bond. Additionally, N-cycloalkyl-D-aldonamides and N-phenyl-D-aldonamides were also synthesized. Of these novel small molecules, two very potent IRI active molecules were discovered: a “disaccharide” possessing an aryl group at the anomeric position with the open-alditol chain of D-galactose linked via an amide bond at C3 and N-phenyl-D-arbonamide. Both of these small molecules were assessed for their ability to cryopreserve hematopoietic stem cells. Unfortunately, the additional of these compounds failed to improved percent cell viabilities as compared to DMSO