Small Molecule Ice Recrystallization Inhibitors Research Articles

C-1007: The design of small molecule ice recrystallization inhibitors for use as cryopreservatives

Ice recrystallization inhibition (IRI) activity is a desirable property for a cryoprotectant. Recently, small molecules having potent IRI activity have been reported by our laboratory ( Chem. Sci. , 2012, 3, 1408). These small molecules are amphiphilic and can form micelles due to their polar carbohydrate-based head and aliphatic tail ( Chem. Sci. , 2012, 3, 1408). We have previously described a correlation between monosaccharide hydrate and IRI activity ( J. Am. Chem. Soc., 2008, 130 (10), 2928) and therefore the effect of adding hydrophobic atoms or functional groups to small molecules on IRI activity was investigated. Fluorine is of particular interest because it possesses what’s known as the ‘hydrophobic hydration effect’, in which water molecules tend to order around fluorine atoms in solution ( ChemBioChem , 2004, 5, 622). Interestingly, the location of a fluorine atom or other hydrophobic functional groups on the aliphatic tail greatly affects the IRI activity of the molecule. During the course of this investigation, several very potent inhibitors of ice recrystallization were discovered and their use as cryoprotectants for Tf-1 α cells, used as a model for stem cells derived from human umbilical cord blood, was explored. Post-thaw cell viability and apoptosis of Tf-1 α cells cryopreserved with these IRI active molecules will be presented.

C-1001: Small molecule inhibitors of ice recrystallization – From design to mechanism of action and applications

Ice recrystallization has been identified as a significant contributor to cellular injury and death during cryopreservation. Unfortunately, many cryopreservation protocols are suboptimal. Thus, interest has arisen in designing novel ice recrystallization inhibitors and using these moleclues as cryoprotectants. Recently, our laboratory has reported several classes of small molecules that have the ability to inhibit ice recrystallization. These classes include amino acid-based organogelators, non-ionic carbohydrate-based amphiphiles (aryl and alkyl alditols) and O-linked alkyl and aryl glycoside derivatives. We have conducted comprehensive structure function studies to optimize the ability of these molecules to inhibit ice recrystallization and also facilitate a more thorough understanding of their mechanism of action. These studies have revealed that long alkyl chains or substituted aryl groups that add “hydrophobicity” to the molecule are essentail for potent inhibition of ice recrystallization and that the relative position of these groups is important. In addition, the choice of counterion can have a dramatic effect on the ability of these compounds to inhibit ice recrystallization. Collectively, our results suggest that a high degree of hydration of these small molecules (and their counterions where appropriate) is a prerequisite for a potent inhibitor of ice recrystallization.?A number of these small molecules described above have been investigated as cryoprotectants and have been found to freeze human red blood cells (RBCs) using low amounts of glycerol. Deglycerolization studies and analysis of RBC recoveries have revealed important insights in the mechanism of action of these small molecule ice recrystallization inhibitors.

067 Small carbohydrate-based molecules as potent ice recrystallization inhibitors and their application in red blood cell cryopreservation

Red blood cell (RBC) preservation from peripheral blood donations in blood banks has ensured the availability of safe blood supplies for transfusions. RBCs are typically stored refrigerated for a maximum of 42 days. However, more prolonged storage times are required for rare blood type donations and currently the only method available for their long-term storage is cryopreservation. Unfortunately, RBC cryopreservation is costly and time-consuming, requiring extensive washing procedures to reduce the high glycerol cryoprotectant concentration from 40–50% to less than 1%. Consequently, improvements in the RBC cryopreservation process are urgently required. A significant contributor to cellular injury and death during cryopreservation is ice recrystallization. Thus, great interest has arisen in designing ice recrystallization inhibitors for use as novel cryoprotectants. Unfortunately, the structural features necessary for ice recrystallization inhibition (IRI) activity are not known. Potent IRI activity was previously limited to large protein and glycoprotein molecules related to biological antifreezes. However, our laboratory recently reported the first potent small molecule ice recrystallization inhibitors that were carbohydrate-based surfactants and hydrogelators. Further investigation into various other small carbohydrate-based molecules has led to the discovery that several substituted oxygen-linked phenolic-glycosides exhibit potent IRI activity. Structure-function studies have identified that key structural features on these phenolic-glycosides are crucial for potent activity. In particular, the activity of these glycosides is highly sensitive to the position of the functional group on the aromatic ring, as well as hydroxyl group stereochemistry and the nature of the anomeric-linkage on the carbohydrate moiety. The cryoprotective ability of various highly IRI active glycosides with RBCs was also assessed to investigate if glycerol concentrations could be significantly reduced during cryopreservation. These results have indicated that their addition to cryoprotective solutions containing lower glycerol concentrations can decrease post-thaw RBC hemolysis. This is significant as a reduction in glycerol concentration can ultimately decrease deglycerolization times and improve post-thaw RBC quality for transfusions. This presentation will discuss the cryopreservation results and the effectiveness of these highly IRI active glycosides as cryo-additives for RBCs, as well as the structure-function results identifying key structural features important for the IRI activity of phenolic-glycosides. Source of funding: This research was supported by Canadian Blood Services (CBS), Canadian Institutes of Health Research (CIHR) and Natural Sciences and Engineering Research Council of Canada (NSERC). Conflict of interest: None declared. rben@uottawa.ca

065 Designing potent inhibitors of Ice recrystallization - from C-linked antifreeze glycoproteins (AFGPs) to small molecules

Cell-based therapeutics has emerged as a critical aspect of modern health care and has shaped new models of care in stem cell and regenerative therapy. Dimethyl sulfoxide and glycerol are the two most common cryoprotectants. Unfortunately, many of the current cryopreservation protocols are suboptimal resulting in poor cell recoveries and/or compromised cellular function post cryopreservation thus limiting applications. One of the most significant causes of decreased cell viability and impaired function during cryopreservation is direct mechanical damage from ice crystals. Preventing this damage using ice recrystallization inhibitors may greatly reduce cellular injury during cryopreservation and enhance the recovery of specialized cellular functions. Antifreeze glycoproteins (AFGPs) are peptide-based structures found in many deep-sea Teleost fish inhabiting sub-zero environments. These compounds prevent the seeding of ice crystals in vivo and ultimately protect these organisms against cryoinjury and death. For the past decade our laboratory has been actively pursuing the rational design and synthesis of functional carbon-linked (C-linked) AFGP analogues possessing custom-tailored antifreeze activity. These compounds are potent inhibitors of ice recrystallization but do not exhibit thermal hysteresis properties making them attractive candidates as cryoprotectants for the preservation of various biological materials. Structure-function studies of these C-linked glycoconjugates have indicated that the most active ice recrystallization inhibitors contain highly hydrated carbohydrate residues. This discovery has lead to the development of several classes of small molecule carbohydrate derivatives that exhibit potent ice recrystallization inhibition activity and may constitute a novel class of cryoprotectants. To date, several different classes of small molecule ice recrystallization inhibitors (IRIs) have been identified including amino acid-based organogelators, non-ionic carbohydrate-based amphiphiles (aryl and alkyl alditols) and O-linked alkyl and aryl glycoside derivatives. The rational design of these compounds, assessment and quantification of their IRI activity will be presented and the key structural features necessary for potent ice recrystallization inhibition activity will be discussed. Finally, the ability of both C-linked AFGP and the small molecule IRIs to function as cryoprotectants will be described. To properly assess this, several cell lines and primary cells have been examined. In addition, we have investigated the cryopreservation of oncolytic viruses (both encapsulated non-encapsulated). Source of funding: NSERC, CIHR, Canadian Blood Services, Green Centre Canada. Conflict of interest: None declared. rben@uottawa.ca

Developing highly active small molecule ice recrystallization inhibitors based upon C-linked antifreeze glycoprotein analogues

Ice recrystallization during cryopreservation results in a significant amount of cellular damage making compounds that exhibit ice recrystallization inhibition (IRI) activity desirable as a novel class of cryoprotectants. Herein, we report a systematic structure–function study on a previously identified IRI active C-linked antifreeze glycoprotein (C-AFGP) analogue (1). These studies indicate that while C-AFGPs containing three tripeptide repeats exhibit weak IRI activity 5.5 μM, a minimum number of four tripeptide repeats is required for potent IRI activity at this concentration. In addition, the galactosyl–ornithine building block 5 exhibited only moderate activity at 22 mM, but IRI activity was significantly increased upon addition of two glycine units to the C-terminal end of the amino acid bearing the C-linked galactopyranose residue. Finally, we report that conjugation of long alkyl chains (octyl, nonyl and decyl) to the C-linked galactosyl moiety of 1 can furnish IRI active small molecules. The “ideal” hydrocarbon chain length was 10 carbons for potent activity in this series of compounds. Longer hydrocarbon chain lengths dramatically decreased solubilities. The results of this study emphasize the importance of hydrophobic moieties for IRI activity and while consistent with previously reported small molecule carbohydrate-based and lysine-based ice recrystallization inhibitors, is the first example where a large IRI active glycoconjugate has been successfully truncated to small molecule IRI active components.

Structurally diverse disaccharide analogs of antifreeze glycoproteins and their ability to inhibit ice recrystallization

The β-d-galactosyl-(1,3)-α-N-acetyl-d-galactosamine disaccharide is present in antifreeze glycoproteins (AFGPs). Analogs of this disaccharide including the β-linked (1,3)-, (1,4)-, and (1,6)-galactosyl-N-acetyl galactosamine and the β-(1,3)-galactosyl-galactoside were synthesized and evaluated for ice recrystallization inhibition (IRI) activity. The results from this study demonstrate that the β-linked-(1,4) disaccharide exhibits more potent IRI activity than the native β-linked-(1,3) disaccharide. The C2 N-acetyl group of the disaccharide does not affect IRI activity but in monosaccharides, the presence of the C2 N-acetyl group decreases IRI activity. The current study will facilitate the design of potent small-molecule ice recrystallization inhibitors.