Ice recrystallization during thawing after cryopreservation results in extensive cellular damage that ultimately leads to cell death and decreased cell viabilities. This is a significant problem particularly with cryopreserved cells utilized in various regenerative medicine therapies. Given the success of these therapies to treat spinal cord injury, cartilage lesions, and cardiacdisease, the development of new and improved cryprotectants that minimize cell damageduring freeze-thawing and improve cell viability post-cryopreservation are urgently required. The current cryopreservative dimethyl sulfoxide, DMSO, is associated with cytotoxicity in clinical settings and is not an optimal cryopreservative. Our laboratory is interested in synthesizing small molecules that possess the property of ice recrystallization inhibition (IRI) activity that can be utilized as cryopreservatives without the cytotoxic effects associated with DMSO. This thesis focuses on the development of small molecule ice recrystallization inhibitors and elucidating the structural features of disaccharides and surfactants that are responsible for potent IRI activity. The first part of this study examines simple disaccharide derivatives mimicking those found in the native AFGP to determine whether disaccharide structure influences IRI activity. Towards this end, the (1,6)-linked AFGP disaccharide analogue was synthesized, assessed for IRI activity using a splat-cooling assay, and compared to the native (1,3)- and (1,4)-linked AFGP disaccharide analogues. The change in linkage was found to have a profound affect on IRI activity. The second part of the study focuses on surfactants and gelators as ice recrystallization inhibitors. Our laboratory has demonstrated that carbohydrate-based hydrogelators can be potent inhibitors of ice recrystallization. While our studies have indicated that a delicate balance between hydrophobic and hydrophilic interactions is crucial for ice recrystallization inhibition (IRI) activity, the essential structural features necessary for potent IRI activity remain unknown. To address this issue, structurally diverse amino acid-based surfactants/gelators, anti-ice nucleating agents, and glycoconjugates were synthesized and assessed for IRI activity. The results indicate that long alkyl chains and increased hydrophobicity are important for potent IRI activity and iii that the position of these alkyl chains is essential. Also, the counterion of these compounds affects the IRI activity and is related to the counterion degree of hydration. These compounds were assessed for their ability to cryopreserve human liver cells (Hep G2) and human bone marrow cells (Tf-1α) in cell-based assays. Additionally, the best IRI assay solution was determined, which involved studying how the salts of the phosphate buffered saline (PBS) solution modulated IRI activity. Finally, small molecule ice recrystallization inhibitors were assessed for their ability to protect the viral vectors vaccinia virus, vesicular stomatitis virus, and herpes simplex-1 virus at various storage conditions. This will aid in developing improved preservation protocols for vaccines and viruses utilized in cancer therapy (oncolytic viruses).

Over the past few decades, there has been an increase in the development of new cellular therapies used for the treatment of various conditions. Thus, the rapid development of therapies requiring transfusion and transplantation of cells has resulted in a need to preserve these cellular therapy products. Cryopreservation is the only currently used method for the long-term storage of cells. The most commonly used cryoprotectants are 10% dimethyl sulfoxide (DMSO) for hematopoietic stem cells (HSCs) and 40% glycerol for red blood cells (RBCs). DMSO fails to protect the functionality of HSCs after cryopreservation and therefore, up to 20% of HSC transplantations fail to engraft. The glycerol in thawed RBC units must be removed to <1% to prevent intravascular hemolysis which is time-consuming. Thus, there is an urgent need to develop improved cryoprotectants for HSCs and RBCs. DMSO and glycerol are unable to control ice recrystallization which is a major source of cellular injury during cryopreservation. Therefore, compounds with the ability to inhibit ice recrystallization could represent a new class of cryoprotectant with a novel mechanism of action. This thesis focuses on the rational design of small-molecule ice recrystallization inhibitors. The key structural attributes required for ice recrystallization inhibition (IRI) activity are investigated. The amphiphilic balance required for IRI activity is explored. Furthermore, two new classes of small-molecule IRIs containing aromatic rings were rationally designed. As a result, several very highly IRI active molecules were discovered. The use of IRIs to improve the cryopreservation of HSCs and RBCs was explored. A number of IRIs improved the post-thaw functionality of HSCs. Supplementation of the current cryoprotectant solution with IRIs resulted in an increase in CFU recovery and frequency of multipotent progenitors. This would reduce the percentage of engraftment failure and allow for a larger proportion of cord blood banks’ inventory to provide an adequate dose for patients requiring transplants. Several IRIs were found to be effective cryoprotectants for RBCs with reduced amounts of glycerol. This could reduce the deglycerolization time for RBCs. These results demonstrate the potential of small-molecule IRIs to improve the current cryopreservation procedures for important cellular therapy products.

Inhibiting the formation of ice is an essential process commercially, industrially, and medically. Compounds that work to stop the formation of ice have historically possessed drawbacks such as toxicity or prohibitively high active concentrations. One class of molecules, ice recrystallization inhibitors, work to reduce the damage caused by the combination of small ice crystals into larger ones. Recent advances made by the Ben lab have identified small molecule carbohydrate analogues that are highly active in the field of ice recrystallization and have potential in the cryopreservation of living tissue. A similar class of molecules, kinetic hydrate inhibitors, work to prevent the formation of another type of ice – gas hydrate. Gas hydrates are formed by the encapsulation of a molecule of a hydrocarbon inside a growing ice crystal. These compounds become problematic in high pressure and low temperature areas where methane is present - such as an oil pipeline. A recent study has highlighted the effects of antifreeze glycoprotein, a biological ice recrystallization inhibitor, in the inhibition of methane clathrates. Connecting these two fields through the synthesis and testing of small molecule ice recrystallization inhibitors in the inhibition of methane hydrates is unprecedented and may lead to a novel class of compounds.

Inhibiting the formation of ice is an essential process commercially, industrially, and medically. Compounds that work to stop the formation of ice have historically possessed drawbacks such as toxicity or prohibitively high active concentrations. One class of molecules, ice recrystallization inhibitors, work to reduce the damage caused by the combination of small ice crystals into larger ones. Recent advances made by the Ben lab have identified small molecule carbohydrate analogues that are highly active in the field of ice recrystallization and have potential in the cryopreservation of living tissue. A similar class of molecules, kinetic hydrate inhibitors, work to prevent the formation of another type of ice – gas hydrate. Gas hydrates are formed by the encapsulation of a molecule of a hydrocarbon inside a growing ice crystal. These compounds become problematic in high pressure and low temperature areas where methane is present - such as an oil pipeline. A recent study has highlighted the effects of antifreeze glycoprotein, a biological ice recrystallization inhibitor, in the inhibition of methane clathrates. Connecting these two fields through the synthesis and testing of small molecule ice recrystallization inhibitors in the inhibition of methane hydrates is unprecedented and may lead to a novel class of compounds.

Inhibiting the formation of ice is an essential process commercially, industrially, and medically. Compounds that work to stop the formation of ice have historically possessed drawbacks such as toxicity or prohibitively high active concentrations. One class of molecules, ice recrystallization inhibitors, work to reduce the damage caused by the combination of small ice crystals into larger ones. Recent advances made by the Ben lab have identified small molecule carbohydrate analogues that are highly active in the field of ice recrystallization and have potential in the cryopreservation of living tissue. A similar class of molecules, kinetic hydrate inhibitors, work to prevent the formation of another type of ice – gas hydrate. Gas hydrates are formed by the encapsulation of a molecule of a hydrocarbon inside a growing ice crystal. These compounds become problematic in high pressure and low temperature areas where methane is present - such as an oil pipeline. A recent study has highlighted the effects of antifreeze glycoprotein, a biological ice recrystallization inhibitor, in the inhibition of methane clathrates. Connecting these two fields through the synthesis and testing of small molecule ice recrystallization inhibitors in the inhibition of methane hydrates is unprecedented and may lead to a novel class of compounds.

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. 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. 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. 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.

With emerging trends of new cellular therapies, the need for quick access to cellular components is necessary. For most applications genetically compatible biological components are essential to prevent adverse immune responses and graft-versus host disease (GVHD). Since these biological components have a limited window to be used, techniques for long-term storage are needed. Cryopreservation is essential for this in the field of biobanking and regenerative medicine to allow for long-term storage of cell products. During this process, ice recrystallization is the major contributor to cell death and decreased cell viability post-thaw. Due to this, controlling ice growth and recrystallization is imperative to increasing cell survival and function. The Ben lab is focused on the synthesis of small molecule, carbohydrate-based cryoprotectants that function as ice recrystallization inhibitors (IRIs). Previously, many IRIs have been synthesized showing varying degrees of ice recrystallization inhibition (IRI). Through the structure-function work, a delicate balance between hydrophobic and hydrophilic portions on the same molecule must be met. These compounds are believed to disrupt hydrogen bonding networks present in the formation of ice, and control ice growth. While numerous types of functional groups on carbohydrate derivatives have been explored, many highly solvated functional groups (amines, sulfates, phosphates, etc.) have not been thoroughly investigated. Highly solvated functional groups should disrupt hydrogen bond networks due to their solvation and in theory, should illicit an IRI response. Sulfate groups have not previously been studied, but are present in several different biological processes, such as immune response and blood coagulation. This suggests that sulfated carbohydrates should be well tolerated biologically. Sulfate groups can also be easily installed on existing IRI active molecules through orthogonal protecting group chemistry. The first part of this thesis is focused on the synthesis and IRI activity of sulfated carbohydrates based upon previously synthesized, IRI active pyranose derivatives. When compared to their parent compounds, most of the sulfated derivatives were less active, but all compounds were incredibly soluble in aqueous media. These derivatives did not show much promise as new IRIs due to the length of their synthesis and reduced IRI activity compared to their parent compounds. The Ben lab has also developed a new class of IRI active carbohydrates: aldonamide derivatives. These compounds are open-chain carbohydrates with an amide bond, arising from the ring opening of a carbohydrate lactone with a substituted amine. While many of these compounds displayed high degrees of IRI activity, many were incredibly insoluble in aqueous systems (many with solubility limits under 50 mM). Since sulfate groups were able to greatly increase solubility with some derivatives retaining IRI activity, installing sulfate groups on existing aldonamide-based IRIs should increase their solubility. Additionally, since many of these derivatives display high degrees of IRI activity, a reduction in IRI activity can be tolerated. Similarly, to the sulfated pyranose derivatives, the presence of a sulfate group reduced the IRI activity compared to the parent compounds in most derivatives. Though some sulfated derivatives possessed a higher degree of IRI activity, all the derivatives experienced a drastic increase in solubility (over 200 mM in PBS). Some of the sulfated aldonamide derivatives were assessed for their ability to protect red blood cells (RBCs) during freezing with reduced glycerol concentrations (15% glycerol), although none of thew tested derivatives showed an improvement over existing IRIs explored by the Ben lab. Since the introduction of sulfate groups to existing IRIs drastically increased solubility in aqueous systems, but resulted in reduced IRI activity in most compounds, focus was switched to the addition of different hydrophilic functional groups. Amino functional groups were briefly explored with galactose-based pyranose IRIs, aldonamide derivatives had not been explored. Amino groups are present on many biological carbohydrates and should be well tolerated biologically. The addition of amino groups to aldonamide derivatives should increase solubility, with the amino derivatives ideally retaining some IRI activity. The amino aldonamide derivatives synthesized had high solubilities (>500 mM in PBS), but did possess lower degrees of IRI activity. Due to the high solubility these derivatives were initially assessed in the cryopreservation of RBCs with reduced glycerol concentrations. Initial experiments showed improvements over current IRIs, and the compounds were assessed in a number of other biological cryopreservation scenarios including articular cartilage, platelets, and hematopoietic stem/progenitor cells (HSPCs). While the compounds showed toxicity in these cell types, more studies need to be conducted for the cryopreservation of RBCs.

Cryopreservation of biological materials has many useful applications and is currently the most effective long term storage method used across a variety of fields. The success of freezing products or biological materials, however, varies because of the process' complexity and related cryo-injuries. One of the primary issues is the ice recrystallization induced development of extracellular and intracellular ice throughout the freezing and thawing process. Ice recrystallization is a significant contributor to freezing damage, ultimately reducing post-thaw viability and function. To address this issue, the Ben laboratory has developed and synthesized a variety of classes of small molecule carbohydrate-based ice recrystallization inhibitors (IRIs). These compounds act as supplements or alternatives to current cryoprotectants, such as trehalose, DMSO, or glycerol, which do not address ice recrystallization and can be cytotoxic. This thesis focuses on the comprehensive chemical property assessment of N-Aryl-β-D-aldonamides and N-Benzyl-β-D-gluconamides, as well as optimization of biopreservation protocols for tissue products and freeze-dried proteins. Utilizing a 5-minute modified splat cooling assay, dose-response curves of five N-Benzyl-β-D-gluconamides were generated. All compounds produced ice recrystallization inhibition active IC50 values comparable to previously investigated active compounds such as, N-Octyl-β-D-gluconamide and N-4-Bromophenyl-β-D-glucopyranoside. Furthermore, validation that the dose-response curves follow a 4-parameter logistic (4PL) or 5PL sigmodal trend depending on symmetry was obtained. In addition, all tested compounds had lower cytotoxicity than N-4-Bromophenyl-β-D-glucopyranoside and higher solubility than N-Octyl-β-D-gluconamide. Overall, N-Benzyl-β-D-gluconamides proved to be a promising class of compounds with the para derivatives being the most IRI active. The second part of this work involved the examination of IRIs' ability to cryopreserve two different biological materials using different biopreservation protocols. The first being proteins and master mix (enzymes and oligonucleotides) during RT-qPCR after the freeze-drying process. The data showed that the IRIs did not interfere and were effective during both the lyophilization and qPCR processes. When compared to most effective concentration of the current industry standard, N-4-Bromophenyl-β-D-glucopyranoside increased the protein activity by ~30%, reducing the number of cycles to reach threshold value. The most significant contribution of this work was the discovery that carbohydrate-based small molecules may be working in more than one mechanism, as both cryoprotectants and lyoprotectants. In addition to proteins, the ability of IRIs to cryopreserve tissue products was investigated. Cell media supplemented with IRIs indicated that they can increase viability and reduce mortality in both cell suspension and single dermal sheets. With N-4-Methylbenzyl-β-D-gluconamide and N-Octyl-β-D-gluconamide being the most effective at reducing the damage associated with freezing and increasing recovery of the cells within the system of a simple one cell type thin tissue matrix.

Cryopresevation is the process of cooling biological materials to low sub-zero temperatures for storage purposes. Numerous medical and technical applications, such as hematopoeitic stem cell transplantation and sperm banking, sometimes require the use of cryopreserved cells. Cryopreservation, however, can induce cell injury and reduce the yields of viable functional cells. Ice recrystallization is a mechanism of cryopreservation injury, but is rarely addressd in strategies to optimize cell cryopreservation. The results from this thesis demonstrate an association between the potency of carbohydrate-mediated ice recrystallization inhibition used in the cryopreservation of umbilical cord blood and recovery of viable non-apoptotic cells and hematopoietic progenitor function. Furthermore, increased numbers of apoptotic cells in hematopoeitic stem cell grafts were associated with reduced hematopoietic function and delayed hematopoietic recovery in patients undergoing blood stem cell transplantation. These findings provide a basis for pursuing further studies assessing ice recrystallization inhibition as a strategy for improving cell cryopreservation.

Cryopresevation is the process of cooling biological materials to low sub-zero temperatures for storage purposes. Numerous medical and technical applications, such as hematopoeitic stem cell transplantation and sperm banking, sometimes require the use of cryopreserved cells. Cryopreservation, however, can induce cell injury and reduce the yields of viable functional cells. Ice recrystallization is a mechanism of cryopreservation injury, but is rarely addressd in strategies to optimize cell cryopreservation. The results from this thesis demonstrate an association between the potency of carbohydrate-mediated ice recrystallization inhibition used in the cryopreservation of umbilical cord blood and recovery of viable non-apoptotic cells and hematopoietic progenitor function. Furthermore, increased numbers of apoptotic cells in hematopoeitic stem cell grafts were associated with reduced hematopoietic function and delayed hematopoietic recovery in patients undergoing blood stem cell transplantation. These findings provide a basis for pursuing further studies assessing ice recrystallization inhibition as a strategy for improving cell cryopreservation.