The goal of our research is to generate cryopreservation agents derived from antifreeze glycoproteins. One postulated mechanism of cell cryo-injury is ice recrystallization. It is known that simple saccharides and cryopreservation agents (DMSO) display ice recrystallization inhibition (IRI). This study assessed the cytotoxicity and cryopreservation ability of these sugars in relation to their IRI. It was determined that compounds with greater IRI have increased cytotoxicity yet confer cryoprotection. To further investigate how structure is affecting IRI activity, several galactopyranoside derivatives were synthesized. A series of deoxy and α-Callyl- deoxy galactopyranoses were prepared. Testing determined that removal of any hydroxyl group removes IRI. 3-deoxy-β-thiophenyl galactose was also synthesized and had surprisingly better IRI than β-thiophenylgalactose. Also, 6-azido galactose had similar IRI to 6-deoxy galactose. Lastly, a series of β- thioalkylgalactosides was synthesized and testing gave contradicting results which suggest that predicting IRI based on hydrophilicity is more complicated than initially hypothesized.

The cryopreservation of cells and tissues requires the addition of a cryoprotectant in order to prevent cellular damage caused by ice. Unfortunately, common cryoprotectants such as DMSO and glycerol exhibit significant toxicity which makes their use unfeasible for many clinical procedures. Our laboratory is interested in the development of alternative, non-toxic cryoprotectants which possess ice recrystallization inhibition (IRI) activity. Potent IRI activity has recently been discovered in certain small molecules, but the structural features required for this process are unclear. Herein we report the development of a library of O-aryl glycosides in order to probe the importance of electron density and hydrophobic moieties for IRI activity. It was found that the degree of electron density at the anomeric oxygen does not correlate with IRI ability in para-substituted aryl glycosides, nor does changing the position of the aryl substituent impart a predictable effect on activity. However, the addition of hydrophobic alkyl or acyl chains was beneficial for IRI activity; generally, increasing chain length was found to correlate with increasing activity. In some instances, an optimal alkyl chain length was identified, after which continued lengthening results in a loss of potency. We conclude from this study that a certain extent of hydrophobic character is beneficial for the IRI activity of aryl glycosides, and that a balance between hydrophobicity and hydrophilicity is required for optimum IRI ability. It is hoped that these findings will aid future efforts towards the rational design of novel cryoprotectants.

The development of effective methods to cryopreserve precious cell types has had tremendous impact on regenerative and transfusion medicine. Hematopoietic stem cell (HSC) transplants from cryopreserved umbilical cord blood (UCB) have been used for regenerative medicine therapies to treat conditions including hematological cancers and immodeficiencies. Red blood cell (RBC) cryopreservation in blood banks extends RBC storage time from 42 days (for hypothermic storage) to 10 years and can overcome shortages in blood supplies from the high demand of RBC transfusions. Currently, the most commonly utilized cryoprotectants are 10% dimethyl sulfoxide (DMSO) for UCB and 40% glycerol for RBCs. DMSO is significantly toxic both to cells and patients upon its infusion. Glycerol must be removed to <1% post-thaw using complicated, time consuming and expensive deglycerolization procedures prior to transfusion to prevent intravascular hemolysis. Thus, there is an urgent need for improvements in cryopreservation processes to reduce/eliminate the use of DMSO and glycerol. Ice recrystallization during cryopreservation is a significant contributor to cellular injury and reduced cell viability. Compounds capable of inhibiting this process are thus highly desirable as novel cryoprotectants to mitigate this damage. The first compounds discovered that were ice recrystallization inhibitors were the biological antifreezes (BAs), consisting of antifreeze proteins and glycoproteins (AFPs and AFGPs). As such, BAs have been explored as potential cryoprotectants, however this has been met with limited success. The thermal hysteresis (TH)activity and ice binding capabilities associated with these compounds can facilitate cellular damage, especially at the temperatures associated with cryopreservation. Consequently, compounds that possess “custom-tailored” antifreeze activity, meaning they exhibit the potent ice recrystallization inhibition (IRI) activity without the ability to bind to ice or exhibit TH activity,are highly desirable for potential use in cryopreservation. This thesis focuses on the rational design of potent ice recrystallization inhibitors and on elucidating important key structural motifs that are essential for potent IRI activity. While particular emphasis in on the development of small molecule IRIs, exploration into structural features that influence the IRI of natural and synthetic BAs and BA analogues is also described as these are some of the most potent inhibitors known to date. Furthermore, this thesis also investigates the use of small molecule IRIs for the cryopreservation of various different cell types to ascertain their potential as novel cryoprotectants to improve upon current cryopreservation protocols, in particular those used for the long-term storage of blood and blood products. Through structure-function studies the influence of (glyco)peptide length, glycosylation and solution structure for the IRI activity of synthetic AFGPs and their analogues is described. This thesis also explores the relationship between IRI, TH and cryopreservation ability of natural AFGPs, AFPs and mutants of AFPs. While these results further demonstrated that BAs are ineffective as cryoprotectants, it revealed the potential influence of ice crystal shape and growth progression on cell survival during cryopreservation. One of the most significant results of this thesis is the discovery of alkyl- and phenolicglycosides as the first small molecule ice recrystallization inhibitors. Prior to this discovery, all reported small molecules exhibited only a weak to moderate ability to inhibit ice recrystallization. To understand how these novel small molecules inhibit this process, structure-function studies were conducted on highly IRI active molecules. These results indicated that key structural features, including the configuration of carbons bearing hydroxyl groups and the configuration of the anomeric center bearing the aglycone, are crucial for potent activity. Furthermore, studies on the phenolic-glycosides determined that the presence of specific substituents and their position on the aryl ring could result in potent activity. Moreover, these studies underscored the sensitivity of IRI activity to structural modifications as simply altering a single atom or functional group on this substituent could be detrimental for activity. Finally, various IRI active small molecules were explored for their cryopreservation potential with different cell types including a human liver cell line (HepG2), HSCs obtained from human UCB, and RBCs obtained from human peripheral blood. A number of phenolic-glycosides were found to be effective cryo-additives for RBC freezing with significantly reduced glycerol concentrations (less than 15%). This is highly significant as it could drastically decrease the deglycerolization processing times that are required when RBCs are cryopreserved with 40% glycerol. Furthermore, it demonstrates the potential for IRI active small molecules as novel cryoprotectants that can improve upon current cryopreservation protocols that are limited in terms of the commonly used cryoprotectants, DMSO and glycerol.

The goal of our research is to generate cryopreservation agents derived from antifreeze glycoproteins. One postulated mechanism of cell cryo-injury is ice recrystallization. It is known that simple saccharides and cryopreservation agents (DMSO) display ice recrystallization inhibition (IRI). This study assessed the cytotoxicity and cryopreservation ability of these sugars in relation to their IRI. It was determined that compounds with greater IRI have increased cytotoxicity yet confer cryoprotection. To further investigate how structure is affecting IRI activity, several galactopyranoside derivatives were synthesized. A series of deoxy and α-Callyl- deoxy galactopyranoses were prepared. Testing determined that removal of any hydroxyl group removes IRI. 3-deoxy-β-thiophenyl galactose was also synthesized and had surprisingly better IRI than β-thiophenylgalactose. Also, 6-azido galactose had similar IRI to 6-deoxy galactose. Lastly, a series of β- thioalkylgalactosides was synthesized and testing gave contradicting results which suggest that predicting IRI based on hydrophilicity is more complicated than initially hypothesized.

Cryopreservation of human red blood cells (RBCs) extends the storage time from 42 days (hypothermic storage limit) to a maximum of 10 years. While this reduces the possibility of RBC shortages in emergency situations, this preservation method is currently limited to individuals with rare blood phenotypes, patients who require autologous blood transfusions, and military applications. Furthermore, cryopreservation is associated with a high degree of cellular damage, which can subsequently reduce the viability of cells post-thaw. The cellular damage incurred upon cryopreservation is primarily attributed to the process of ice recrystallization. To reduce the degree of cellular damage, cryoprotective agents (CPAs) are used. Currently, 10 % dimethyl sulphoxide (DMSO) and 40 % glycerol are used for the cryopreservation of hematopoietic stem cells (HSCs) and human RBCs respectively. Unfortunately, these CPAs do not provide protection against ice recrystallization. The biological antifreezes (BAs) consisting of antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) were identified as the first inhibitors of ice recrystallization. Consequently, the Ben laboratory is interested in synthesizing small molecule carbohydrate-based inhibitors of ice recrystallization that can be used as an alternative to glycerol or DMSO for the cryopreservation of various cell types. Therefore, this thesis focuses on elucidating important structural features of carbohydrate-based derivatives that are responsible for IRI activity. The first part of this study examines the importance of the anomeric oxygen atom of aryl glycosides for IRI activity. Our laboratory previously demonstrated that the O-linked aryl glycosides are effective inhibitors of ice recrystallization. However, the influence of stereoelectronic effects at the C1 position of aryl glycosides on IRI activity has not been investigated. As a result, N- and S-linked aryl glycosides were synthesized in this study and their IRI activities were compared to that of the O-linked aryl glycosides. These results suggest that a stronger exo-anomeric effect exhibited by the C1 nitrogen derivatives reduces the IRI activity of aryl glycosides. The second part of this study focuses on the synthesis of AFGP disaccharide analogs. While the β-(1,3) glycosidic linkage found in native AFGP-8 was previously assessed for its influence on IRI activity, an extensive structure-function analysis of AFGP disaccharide analogs has not yet been performed. As a result, an AFGP disaccharide analog was designed whereby a para-methoxyphenyl (PMP) substituent was incorporated. This was done to assess whether the PMP substituent could enhance the lack of IRI activity exhibited previously with AFGP disaccharide analogs. Although the synthesis of this disaccharide target was not completed, a number of advantageous developments have been made regarding the glycosylation of N-acetyl-D-glycosamine derivatives. In addition, the PMP-GlcNAc intermediate encountered in disaccharide synthesis was assessed for its IRI activity, confirming that the acetamido (NHAc) function is not required for IRI activity.

The cryopreservation of cells and tissues requires the addition of a cryoprotectant in order to prevent cellular damage caused by ice. Unfortunately, common cryoprotectants such as DMSO and glycerol exhibit significant toxicity which makes their use unfeasible for many clinical procedures. Our laboratory is interested in the development of alternative, non-toxic cryoprotectants which possess ice recrystallization inhibition (IRI) activity. Potent IRI activity has recently been discovered in certain small molecules, but the structural features required for this process are unclear. Herein we report the development of a library of O-aryl glycosides in order to probe the importance of electron density and hydrophobic moieties for IRI activity. It was found that the degree of electron density at the anomeric oxygen does not correlate with IRI ability in para-substituted aryl glycosides, nor does changing the position of the aryl substituent impart a predictable effect on activity. However, the addition of hydrophobic alkyl or acyl chains was beneficial for IRI activity; generally, increasing chain length was found to correlate with increasing activity. In some instances, an optimal alkyl chain length was identified, after which continued lengthening results in a loss of potency. We conclude from this study that a certain extent of hydrophobic character is beneficial for the IRI activity of aryl glycosides, and that a balance between hydrophobicity and hydrophilicity is required for optimum IRI ability. It is hoped that these findings will aid future efforts towards the rational design of novel cryoprotectants.

The development of effective methods to cryopreserve precious cell types has had tremendous impact on regenerative and transfusion medicine. Hematopoietic stem cell (HSC) transplants from cryopreserved umbilical cord blood (UCB) have been used for regenerative medicine therapies to treat conditions including hematological cancers and immodeficiencies. Red blood cell (RBC) cryopreservation in blood banks extends RBC storage time from 42 days (for hypothermic storage) to 10 years and can overcome shortages in blood supplies from the high demand of RBC transfusions. Currently, the most commonly utilized cryoprotectants are 10% dimethyl sulfoxide (DMSO) for UCB and 40% glycerol for RBCs. DMSO is significantly toxic both to cells and patients upon its infusion. Glycerol must be removed to <1% post-thaw using complicated, time consuming and expensive deglycerolization procedures prior to transfusion to prevent intravascular hemolysis. Thus, there is an urgent need for improvements in cryopreservation processes to reduce/eliminate the use of DMSO and glycerol. Ice recrystallization during cryopreservation is a significant contributor to cellular injury and reduced cell viability. Compounds capable of inhibiting this process are thus highly desirable as novel cryoprotectants to mitigate this damage. The first compounds discovered that were ice recrystallization inhibitors were the biological antifreezes (BAs), consisting of antifreeze proteins and glycoproteins (AFPs and AFGPs). As such, BAs have been explored as potential cryoprotectants, however this has been met with limited success. The thermal hysteresis (TH)activity and ice binding capabilities associated with these compounds can facilitate cellular damage, especially at the temperatures associated with cryopreservation. Consequently, compounds that possess “custom-tailored” antifreeze activity, meaning they exhibit the potent ice recrystallization inhibition (IRI) activity without the ability to bind to ice or exhibit TH activity,are highly desirable for potential use in cryopreservation. This thesis focuses on the rational design of potent ice recrystallization inhibitors and on elucidating important key structural motifs that are essential for potent IRI activity. While particular emphasis in on the development of small molecule IRIs, exploration into structural features that influence the IRI of natural and synthetic BAs and BA analogues is also described as these are some of the most potent inhibitors known to date. Furthermore, this thesis also investigates the use of small molecule IRIs for the cryopreservation of various different cell types to ascertain their potential as novel cryoprotectants to improve upon current cryopreservation protocols, in particular those used for the long-term storage of blood and blood products. Through structure-function studies the influence of (glyco)peptide length, glycosylation and solution structure for the IRI activity of synthetic AFGPs and their analogues is described. This thesis also explores the relationship between IRI, TH and cryopreservation ability of natural AFGPs, AFPs and mutants of AFPs. While these results further demonstrated that BAs are ineffective as cryoprotectants, it revealed the potential influence of ice crystal shape and growth progression on cell survival during cryopreservation. One of the most significant results of this thesis is the discovery of alkyl- and phenolicglycosides as the first small molecule ice recrystallization inhibitors. Prior to this discovery, all reported small molecules exhibited only a weak to moderate ability to inhibit ice recrystallization. To understand how these novel small molecules inhibit this process, structure-function studies were conducted on highly IRI active molecules. These results indicated that key structural features, including the configuration of carbons bearing hydroxyl groups and the configuration of the anomeric center bearing the aglycone, are crucial for potent activity. Furthermore, studies on the phenolic-glycosides determined that the presence of specific substituents and their position on the aryl ring could result in potent activity. Moreover, these studies underscored the sensitivity of IRI activity to structural modifications as simply altering a single atom or functional group on this substituent could be detrimental for activity. Finally, various IRI active small molecules were explored for their cryopreservation potential with different cell types including a human liver cell line (HepG2), HSCs obtained from human UCB, and RBCs obtained from human peripheral blood. A number of phenolic-glycosides were found to be effective cryo-additives for RBC freezing with significantly reduced glycerol concentrations (less than 15%). This is highly significant as it could drastically decrease the deglycerolization processing times that are required when RBCs are cryopreserved with 40% glycerol. Furthermore, it demonstrates the potential for IRI active small molecules as novel cryoprotectants that can improve upon current cryopreservation protocols that are limited in terms of the commonly used cryoprotectants, DMSO and glycerol.

No description available.

Given the rising demand for diverse cell types in regenerative and transfusion medicines, such as human induced pluripotent stem cell-derived neurons (iPSC-Ns), human T/chimeric antigen receptor (CAR) T cells, and human natural killer (NK) cells, the ability to cryopreserve cells has become increasingly important. In regenerative medicine, iPSC-Ns are powerful tools for treating and modelling neurodegenerative diseases. Moreover, transplants/transfusions of T/CAR T cells or NK cells offer promising treatment for numerous types of tumors, such as leukemia and multiple myeloma. Cryopreservation of cells at sub-zero temperatures (-80 to -196 °C) allows for the development of master cell banks that can be used for clinical applications. Conventional cryoprotective agents (CPAs), such as dimethylsulfoxide (DMSO) and glycerol, are utilized to protect cells from cryoinjuries associated with the freezing process. However, the use of high concentrations of DMSO (i.e., 10 to 20%) has been shown to be accompanied with toxic effects on patients receiving cell therapies if it is not removed or diluted prior to transfusion. Moreover, DMSO does not prevent the occurrence of the cryoinjury associated with ice recrystallization, which is one of the major causes of cell death/damage during cryopreservation. As a result, there is a surge of attention toward developing new non-toxic cryo-additives that inhibit ice recrystallization during cryopreservation to permit future advancement in regenerative and transfusion medicines. Moreover, the use of ice recrystallization inhibitors (IRIs) as novel CPAs has become a promising strategy to improve cell viability and function post-thaw. The Ben laboratory heavily invested in synthesizing several classes of carbohydrate-based small molecule IRIs (i.e., O-linked alkyl and aryl glycosides, and N-aryl-D-gluconamides), and studying the correlation between their IRI activity and molecular properties, such as polar surface area to molecular surface area (PSA/MSA) ratio. Moreover, compounds that belong to the O-linked aryl glycosides and N-aryl-D-gluconamides classes of IRIs have been shown to enhance the viability and functionality of red blood cells (RBCs), hematopoietic stem cells (HSCs), and induced pluripotent stem cells (iPSCs) after thawing. Part of the research presented throughout this thesis focuses on structure-activity relationship (SAR) studies of alkyl pyranoses with modified alkyl chain lengths to explore any correlations between the IRI activity and the net polarity (i.e., PSA/MSA ratio) of the IRI candidates. O- and C-linked alkyl pyranose derivatives with different alkyl chain lengths were synthesized and their IRI activity was assessed using the modified splat cooling assay. While the IRI activity of the O- and C-linked alkyl glucosides did differ as the length of the alkyl chain increased, no correlation between the PSA/MSA ratios and their IRI activity was observed. In addition, this work allowed for investigation into the effect of the type of the glycosidic bond (i.e., C-O and C-C bonds) at the anomeric position, on the IRI activity of the different compounds. The O-linked alkyl glucosides appeared to be more IRI active than the C-linked compounds, suggesting the nature of the glycosidic bond is important for IRI activity. The second part of the research presented in this thesis focuses on examining the potential for IRIs to cryopreserve iPSC-Ns, T/CAR T cells, and NK cells. 2-fluorophenyl-D-gluconamides (2FA), which is one of the most active IRIs from the N-aryl-Dgluconamides, has shown promising results in maintaining a high number of viable and functional HSCs and iPSCs post-thaw, and therefore it was employed in the cryopreservation protocol of iPSC-Ns, human-derived T/CAR T cells, and human-derived NK cells. The efficacy of the cryopreservation protocol being constructed was evaluated by assessing the post-thaw viability and recovery rate, as well as the functionality of iPSCNs, T/CAR T cells, and NK cells post-thaw. These studies showed that protecting against ice recrystallization during cryopreservation with IRIs increases the number of viable and functional iPSC-Ns, and T/CAR T cells. It was also observed that employing IRI technology in the cryopreservation protocol of NK cells does not compromise their functionality compared to fresh, non-frozen NK cells. Overall, inhibition of ice recrystallization using IRIs appeared to enhance the cryopreservation outcomes of the different cell types, which will allow for the development of off-the-shelf cell therapy products and improvement of the delivery of efficacious cell products to clinics and hospitals.