**6. Inhibitors of ice recrystallization**

growth inhibition of BAs. [153-156] Regardless of these alternate mechanisms, sufficient data exists to suggest an irreversible adsorption-inhibition mechanism, and consequently this

It should be emphasized that the ability to bind to ice is believed to be a property unique to BAs. However, it has been reported that polyvinyl alcohol (PVA) can bind to ice and exhibit a small degree of thermal hysteresis. [157] It was originally proposed that adsorption of BAs to the surface of ice occurred through the hydrogen bonding of hydrophilic groups to the oxygen atoms in the ice lattice. [12,158] However, this is contradictory to the current mecha‐ nism of action for AFPs where the importance of hydrogen bonding between polar residues and ice has been questioned. Alternatively, it has been demonstrated that entropic and enthalpic contributions from hydrophobic residues are crucial for ice binding. [159-161] The importance of hydrophobic residues has been validated with a number of different AFPs through site-specific mutagenesis studies, [82,159,162,163] and in general it is believed that the ice-binding site of these AFPs is hydrophobic and has a discrete complementarity with the

In contrast to AFPs, the current hypothesis of how AFGPs bind to ice involves hydrogen bonding between the hydroxyl groups of the sugars and the ice lattice. [137] A landmark study conducted by Nishimura and co-workers investigated the key structural features of AFGPs that were crucial for ice binding and TH activity. [166] In this study it was reported that three key motifs were required for TH activity (shown in Figure 8): 1) the *N*-acetyl group at the C2 position of the galactosamine; 2) the α-configuration of the *O*-glycosidic linkage between the disaccharide and the peptide chain; 3) the γ-methyl group of the threonyl residue. In addition, the TH activity of homogenous AFGPs is dependent upon the length of the glycoprotein

**Figure 8.** Important structural motifs on AFGPs for TH activity as determined by Nishimura and co-workers. [166]

Despite the tremendous number of structure-function studies conducted on AFPs and AFGPs over the last three decades, in all cases only TH activity has been assessed and correlated to structural modifications. The ability of these analogues to inhibit ice recrystallization has not been assessed, and consequently the structural features necessary for potent ice recrystalliza‐ tion inhibition (IRI) activity are not known. This is unfortunate as IRI activity is a highly

model is the generally accepted mechanism by which BAs exhibit TH activity.

planes of ice to which it binds. [82,162-165]

188 Recent Developments in the Study of Recrystallization

segment. [166,167]

Biological antifreezes are excellent inhibitors of ice recrystallization. However, as stated in the previous section, the dynamic ice shaping (DIS) capabilities prohibits their use in applications where ice recrystallization inhibition (IRI) activity is highly desirable. Thus, the purpose of the following section will be to summarize the progress towards designing molecules that exhibit the ability to inhibit ice recrystallization without the ability to bind to ice, and on understanding the key structural features that are important for the IRI activity exhibited by these molecules.

## **6.1. Peptide and glycopeptide analogues of biological antifreezes as ice recrystallization inhibitors**

One of the first studies that examined ice recrystallization inhibition (IRI) activity of peptides and conventional polymers was conducted by Knight *et al.* in 1995. [41] In this study, a type I winter flounder antifreeze protein and six analogues of this protein were investigated for their ability to inhibit ice recrystallization, along with four polypeptides and three polymers including polyvinyl alcohol (PVA). One of the conclusions from this study was that all analogues of the antifreeze protein were completely IRI inactive in 0.1% and 0.5% NaCl solutions, a result that correlated with the reduced TH activity in comparison to the native AFP exhibited by these analogues. [41,171] It was also reported that poly-L-histidine, poly-Lhydroxyproline and PVA exhibited IRI activity at concentrations less than 1 mg/mL in pure water, whereas poly-L-aspartic acid, poly-L-asparagine, polyacrylic acid and polyvinylpyr‐ rolidone were inactive. These polypeptides and polymers were not assessed for IRI activity in NaCl solutions.

This study ultimately suggested there was a correlation between TH and IRI activity in the type I AFP. [41,171] While it is well known that biological antifreezes exhibit both types of antifreeze activity, the relationship between TH and IRI has been debated throughout the literature. It was previously suggested that these two properties were directly correlated and derived from the ability of BAs to bind to ice. [139,153] In contrast, it has been suggested there is little or no correlation between TH and IRI as some plant AFPs typically exhibit a low degree of TH activity but a high degree of IRI activity. [119,120] Furthermore, the elevated TH activity exhibited by hyperactive insect AFPs is often not accompanied by highly potent IRI activity. [141] To date, few studies have emerged examining the relationship between TH and IRI activity in native BAs, and those that have, report IRI activity using methods other than the traditional splat-cooling assay, [141] making it difficult to ascertain definitive conclusions about the correlation between TH and IRI.

tible to hydrolysis under basic or acidic conditions. The first of these analogues was reported in 2003 (shown in Figure 10). [172] In comparison to AFGPs, the terminal galactose unit and the *N*-acetyl group were removed leaving only an α-D- galactosyl unit that was conjugated to lysine residues. Lysine was used due to its structural similarity to an arginine residue, which was occasionally found in native AFGPs (see section 5.1). In addition, the alanine residues present in AFGPs were substituted with glycine residues to avoid racemization encountered during solid-phase synthesis. [172,173] The monomer tripeptide unit (**7**) and the analogue with three repeating tripeptide units (**8**) did not exhibit IRI activity. However, the analogues with six and nine repeating tripeptide units (derivatives **9** and **10**, respectively) were both moder‐ ately IRI active. Derivatives **9** and **10** were also assessed for TH activity and both exhibited a

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small TH gap of 0.06 °C and induced the formation of hexagonal shaped ice crystals.

**Figure 10.** Structure of first-generation lysine-based *C*-linked AFGP analogues reported by Ben. [172]

exhibited potent IRI activity with little or no measureable TH activity.

The Ben laboratory has published two other *C*-linked AFGP analogues that exhibit potent IRI activity. These are derivatives **11** and **12** (Figure 11). Derivative **11** contains four tripeptide repeats, in which a *C*-linked galactosyl unit is incorporated. [57] Derivative **12** also contains four tripeptide repeats, and is structurally similar to lysine derivatives **8-10**, however the *C*linked α-D- galactosyl unit is conjugated to an ornithine residue. [58] Both of these derivatives exhibited potent IRI activity at 5.5 *μ*M and their activity was similar to that exhibited by AFGP-8 at 5.5 *μ*M. Unlike AFGP-8, neither of these derivatives exhibited TH activity and while **12** exhibited very weak dynamic ice shaping, [58] **11** did not exhibit any ice shaping capabilities. [57] This suggested that the exhibited IRI activity was not likely due to ice binding. These analogues were the first examples where the two properties of biological antifreezes, TH and IRI activity, were decoupled from each other. Additionally, these *C*-linked AFGP analogues were the first compounds that possessed "custom-tailored" antifreeze activity, meaning they

Following the discovery of the two novel synthetic ice recrystallization inhibitors **11** and **12**, two studies have been reported that identify the structural features necessary for the potent IRI activity of these *C*-linked analogues. The first structure-function study was conducted on

Payne and co-workers recently published a study in 2012 examining the correlation of glycopeptide/glycoprotein mass on both TH and IRI activity for a range of homogeneous synthetic AFGPs (synAFGPs). [167] A native chemical ligation-desulfurization approach was used for the first convergent synthesis of homogenous synAFGPs that ranged in molecular mass from 1.2 – 19.5 kDa (compounds **1**-**6**, Figure 9). Increasing the length of the glycopeptide to eight and twelve tripeptide repeats (synAFGP8 and synAFGP12, **3** and **4**) increased TH and IRI activity. However, increasing the number of tripeptide repeats to 16 (synAFGP16, **5**) led to reduced TH and IRI activity. Additional elongation of the glycopeptide to 32 tripeptide repeats (synAFGP32, **6**) restored the potent TH and IRI activities exhibited by these glycopeptides. Interestingly, while synAFGP16 (**5**) exhibited less TH activity than synAFGP8 (**3**), both had similar IRI activities. Furthermore, while synAFGP12 (**4**) and synAFGP32 (**6**) exhibited similar TH and IRI activities and were three times more IRI active than synAFGP8 (**3**) and syn‐ AFGP16 (**5**), they twice as TH active than synAFGP8 and four times as TH active as synAFGP16. These results support the hypothesis that the two types of antifreeze activities may not be as closely correlated as previously thought as the magnitude of change in TH activity was not reflected in IRI activity with these homogenous synAFGPs. While further work is still required in this area to verify this hypothesis, studies on synthetic structural analogues of AFGPs have shown it is possible to decouple the two types of antifreeze activities from each other, resulting in compounds that exhibit "custom-tailored" antifreeze activity and are only IRI active and not TH active. [57,58]

**Figure 9.** Structures of homogeneous synthetic AFGPs (synAFGPs) reported by Payne and co-workers. [167]

Most of the peptide and glycopeptides that have been assessed for IRI activity have been synthetic structural analogues of AFGPs. The Ben laboratory published the first series of analogues with dramatic structural modifications relative to the AFGP structure, and these analogues maintained the potent IRI activity exhibited by AFGP-8 at equimolar concentrations but did not exhibit TH activity. These analogues were carbon-linked or *C*-linked analogues, and consequently did not possess the *O*-glycosidic linkage found in AFGPs which is suscep‐ tible to hydrolysis under basic or acidic conditions. The first of these analogues was reported in 2003 (shown in Figure 10). [172] In comparison to AFGPs, the terminal galactose unit and the *N*-acetyl group were removed leaving only an α-D- galactosyl unit that was conjugated to lysine residues. Lysine was used due to its structural similarity to an arginine residue, which was occasionally found in native AFGPs (see section 5.1). In addition, the alanine residues present in AFGPs were substituted with glycine residues to avoid racemization encountered during solid-phase synthesis. [172,173] The monomer tripeptide unit (**7**) and the analogue with three repeating tripeptide units (**8**) did not exhibit IRI activity. However, the analogues with six and nine repeating tripeptide units (derivatives **9** and **10**, respectively) were both moder‐ ately IRI active. Derivatives **9** and **10** were also assessed for TH activity and both exhibited a small TH gap of 0.06 °C and induced the formation of hexagonal shaped ice crystals.

activity in native BAs, and those that have, report IRI activity using methods other than the traditional splat-cooling assay, [141] making it difficult to ascertain definitive conclusions

Payne and co-workers recently published a study in 2012 examining the correlation of glycopeptide/glycoprotein mass on both TH and IRI activity for a range of homogeneous synthetic AFGPs (synAFGPs). [167] A native chemical ligation-desulfurization approach was used for the first convergent synthesis of homogenous synAFGPs that ranged in molecular mass from 1.2 – 19.5 kDa (compounds **1**-**6**, Figure 9). Increasing the length of the glycopeptide to eight and twelve tripeptide repeats (synAFGP8 and synAFGP12, **3** and **4**) increased TH and IRI activity. However, increasing the number of tripeptide repeats to 16 (synAFGP16, **5**) led to reduced TH and IRI activity. Additional elongation of the glycopeptide to 32 tripeptide repeats (synAFGP32, **6**) restored the potent TH and IRI activities exhibited by these glycopeptides. Interestingly, while synAFGP16 (**5**) exhibited less TH activity than synAFGP8 (**3**), both had similar IRI activities. Furthermore, while synAFGP12 (**4**) and synAFGP32 (**6**) exhibited similar TH and IRI activities and were three times more IRI active than synAFGP8 (**3**) and syn‐ AFGP16 (**5**), they twice as TH active than synAFGP8 and four times as TH active as synAFGP16. These results support the hypothesis that the two types of antifreeze activities may not be as closely correlated as previously thought as the magnitude of change in TH activity was not reflected in IRI activity with these homogenous synAFGPs. While further work is still required in this area to verify this hypothesis, studies on synthetic structural analogues of AFGPs have shown it is possible to decouple the two types of antifreeze activities from each other, resulting in compounds that exhibit "custom-tailored" antifreeze activity and are only IRI active and

**Figure 9.** Structures of homogeneous synthetic AFGPs (synAFGPs) reported by Payne and co-workers. [167]

Most of the peptide and glycopeptides that have been assessed for IRI activity have been synthetic structural analogues of AFGPs. The Ben laboratory published the first series of analogues with dramatic structural modifications relative to the AFGP structure, and these analogues maintained the potent IRI activity exhibited by AFGP-8 at equimolar concentrations but did not exhibit TH activity. These analogues were carbon-linked or *C*-linked analogues, and consequently did not possess the *O*-glycosidic linkage found in AFGPs which is suscep‐

about the correlation between TH and IRI.

190 Recent Developments in the Study of Recrystallization

not TH active. [57,58]

**Figure 10.** Structure of first-generation lysine-based *C*-linked AFGP analogues reported by Ben. [172]

The Ben laboratory has published two other *C*-linked AFGP analogues that exhibit potent IRI activity. These are derivatives **11** and **12** (Figure 11). Derivative **11** contains four tripeptide repeats, in which a *C*-linked galactosyl unit is incorporated. [57] Derivative **12** also contains four tripeptide repeats, and is structurally similar to lysine derivatives **8-10**, however the *C*linked α-D- galactosyl unit is conjugated to an ornithine residue. [58] Both of these derivatives exhibited potent IRI activity at 5.5 *μ*M and their activity was similar to that exhibited by AFGP-8 at 5.5 *μ*M. Unlike AFGP-8, neither of these derivatives exhibited TH activity and while **12** exhibited very weak dynamic ice shaping, [58] **11** did not exhibit any ice shaping capabilities. [57] This suggested that the exhibited IRI activity was not likely due to ice binding. These analogues were the first examples where the two properties of biological antifreezes, TH and IRI activity, were decoupled from each other. Additionally, these *C*-linked AFGP analogues were the first compounds that possessed "custom-tailored" antifreeze activity, meaning they exhibited potent IRI activity with little or no measureable TH activity.

Following the discovery of the two novel synthetic ice recrystallization inhibitors **11** and **12**, two studies have been reported that identify the structural features necessary for the potent IRI activity of these *C*-linked analogues. The first structure-function study was conducted on

**Figure 11.** Structures of potently IRI active *C*-linked AFGP analogues **11** and **12** reported by the Ben laboratory. [57,58] Analogues **11** and **12** are the first compounds reported to exhibit "custom-tailored" antifreeze activity, mean‐ ing they exhibit potent IRI activity but not TH activity.

between the carbohydrate and peptide backbone of derivative **11** was increased such that the side chain linking the carbohydrate to the backbone was two, three or four carbons in length (analogues **11**, **16** and **17**, respectively, Figure 13). [57] The distance between the carbohydrate and peptide backbone of derivative **12** was both increased and decreased such that the side chain linking the carbohydrate to the backbone was a total of four, five, six or seven atoms in length (analogues **18**, **19**, **12** and **20**, respectively, Figure 13). [58,178] All of the analogues in which the side chain lengths were modified failed to exhibit IRI activity. These results indicated that the optimal length of linker between the carbohydrate and peptide backbone is two carbons for analogue **11** and six atoms for analogue **12**. [57,178] Molecular dynamic simulations indicated that **12** adopted a unique conformation in solution that was distinctly different than analogues **18-20**. [178] While **18-20** were found to adopt a conformation in which the carbo‐ hydrate moiety was extended away from the polypeptide backbone, the side chain of **12** was folded back on itself. It was speculated this fold formed a hydrophobic "pocket" between the

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**Figure 12.** Structures of *C*-linked AFGP analogues containing various monosaccharide moieties. [58]

In addition to *C*-linked AFGP analogues, other synthetic variants of AFGPs have recently been assessed for their ability to inhibit ice recrystallization. In 2010, Sewald and co-workers synthesized analogues of AFGP-8 in which alanine residues were replaced with proline residues and the native disaccharide was replaced with the monosaccharide α-*N*-acetyl-Dgalactosamine (Figure 14). [179] It was reported that the glycopeptide analogues containing tripeptide repeats of (Ala-Ala-Thr(GalNHAc))*n* were found to exhibit IRI activity (compounds **21**-**23**). This activity was dependent upon the length of the glycopeptide, and the compound with five tripeptide repeats (**23**) was found to be the most active at a lower concentration (12.5 *μ*M) in comparison to the compound with three tripeptide repeats (**21**) which was active at a much higher concentration (0.8 mM). AFGP analogues **21**-**23** were found to induce hexagonal ice crystal shaping, suggesting that they are interacting with the ice lattice, however the TH

carbohydrate and the peptide, resulting in potent IRI activity.

*C*-linked AFGP analogue **12** and examined the importance of the carbohydrate moiety. [58] The galactosyl moiety of **12** was substituted with three other monosaccharides: glucose, mannose and talose (analogues **13**, **14** and **15**, respectively, Figure 12). It was found that replacing the galactosyl unit with other monosaccharides was highly detrimental for IRI activity. The glucose analogue **13** exhibited weak activity, whereas the mannose and talose analogues (**14** and **15**) were inactive. The results showed that the stereochemical relationship of the hydroxyl groups on the carbohydrate moiety on the polypeptide has a direct affect upon IRI activity. The stereochemical relationship of the hydroxyl groups on simple carbohydrates (mono- and disaccharides) is known to influence the hydration of carbohydrates. [174-176] This lead to the observation that carbohydrate hydration was important for IRI activity. [58] A more detailed discussion of carbohydrate hydration and its influence on IRI activity is provided in section 6.3 of this chapter. Briefly, carbohydrate hydration influences IRI activity by altering the ordering of bulk-water based on the compatibility of the carbohydrate within the three-dimensional hydrogen-bonded network of water. [58,177] The hydration of a carbohydrate is related to the compatibility of the sugar with the three-dimensional hydrogenbond network of water. [174-176] Of the monosaccharides assessed, talose is the most com‐ patible and is thought to have the best "fit" into this hydrogen-bond network, whereas galactose is the least compatible and has the worse "fit". It was hypothesized that a poorer "fit" of the carbohydrate into the hydrogen-bond network of bulk water resulted in a more disordered bulk water layer between the semi-ordered quasi-liquid layer and ordered ice crystal layer. Consequently, transferring water molecules from a more disordered bulk water layer to an ordered layer was energetically unfavorable. Thus, carbohydrates that are highly hydrated resulted in greater IRI activity. [177] While the overall hydration of the *C*-linked glycoconjugates **12**-**15** is not known, having a more highly hydrated carbohydrate moiety conjugated on the glycopeptide (ie. galactose) was significantly better for IRI activity than a less hydrated carbohydrate moiety. [58]

The second structure-function study examined how the distance between the galactosyl moiety and the polypeptide backbone influenced IRI activity. In this study, the distance

**Figure 12.** Structures of *C*-linked AFGP analogues containing various monosaccharide moieties. [58]

*C*-linked AFGP analogue **12** and examined the importance of the carbohydrate moiety. [58] The galactosyl moiety of **12** was substituted with three other monosaccharides: glucose, mannose and talose (analogues **13**, **14** and **15**, respectively, Figure 12). It was found that replacing the galactosyl unit with other monosaccharides was highly detrimental for IRI activity. The glucose analogue **13** exhibited weak activity, whereas the mannose and talose analogues (**14** and **15**) were inactive. The results showed that the stereochemical relationship of the hydroxyl groups on the carbohydrate moiety on the polypeptide has a direct affect upon IRI activity. The stereochemical relationship of the hydroxyl groups on simple carbohydrates (mono- and disaccharides) is known to influence the hydration of carbohydrates. [174-176] This lead to the observation that carbohydrate hydration was important for IRI activity. [58] A more detailed discussion of carbohydrate hydration and its influence on IRI activity is provided in section 6.3 of this chapter. Briefly, carbohydrate hydration influences IRI activity by altering the ordering of bulk-water based on the compatibility of the carbohydrate within the three-dimensional hydrogen-bonded network of water. [58,177] The hydration of a carbohydrate is related to the compatibility of the sugar with the three-dimensional hydrogenbond network of water. [174-176] Of the monosaccharides assessed, talose is the most com‐ patible and is thought to have the best "fit" into this hydrogen-bond network, whereas galactose is the least compatible and has the worse "fit". It was hypothesized that a poorer "fit" of the carbohydrate into the hydrogen-bond network of bulk water resulted in a more disordered bulk water layer between the semi-ordered quasi-liquid layer and ordered ice crystal layer. Consequently, transferring water molecules from a more disordered bulk water layer to an ordered layer was energetically unfavorable. Thus, carbohydrates that are highly hydrated resulted in greater IRI activity. [177] While the overall hydration of the *C*-linked glycoconjugates **12**-**15** is not known, having a more highly hydrated carbohydrate moiety conjugated on the glycopeptide (ie. galactose) was significantly better for IRI activity than a

**Figure 11.** Structures of potently IRI active *C*-linked AFGP analogues **11** and **12** reported by the Ben laboratory. [57,58] Analogues **11** and **12** are the first compounds reported to exhibit "custom-tailored" antifreeze activity, mean‐

The second structure-function study examined how the distance between the galactosyl moiety and the polypeptide backbone influenced IRI activity. In this study, the distance

less hydrated carbohydrate moiety. [58]

ing they exhibit potent IRI activity but not TH activity.

192 Recent Developments in the Study of Recrystallization

between the carbohydrate and peptide backbone of derivative **11** was increased such that the side chain linking the carbohydrate to the backbone was two, three or four carbons in length (analogues **11**, **16** and **17**, respectively, Figure 13). [57] The distance between the carbohydrate and peptide backbone of derivative **12** was both increased and decreased such that the side chain linking the carbohydrate to the backbone was a total of four, five, six or seven atoms in length (analogues **18**, **19**, **12** and **20**, respectively, Figure 13). [58,178] All of the analogues in which the side chain lengths were modified failed to exhibit IRI activity. These results indicated that the optimal length of linker between the carbohydrate and peptide backbone is two carbons for analogue **11** and six atoms for analogue **12**. [57,178] Molecular dynamic simulations indicated that **12** adopted a unique conformation in solution that was distinctly different than analogues **18-20**. [178] While **18-20** were found to adopt a conformation in which the carbo‐ hydrate moiety was extended away from the polypeptide backbone, the side chain of **12** was folded back on itself. It was speculated this fold formed a hydrophobic "pocket" between the carbohydrate and the peptide, resulting in potent IRI activity.

In addition to *C*-linked AFGP analogues, other synthetic variants of AFGPs have recently been assessed for their ability to inhibit ice recrystallization. In 2010, Sewald and co-workers synthesized analogues of AFGP-8 in which alanine residues were replaced with proline residues and the native disaccharide was replaced with the monosaccharide α-*N*-acetyl-Dgalactosamine (Figure 14). [179] It was reported that the glycopeptide analogues containing tripeptide repeats of (Ala-Ala-Thr(GalNHAc))*n* were found to exhibit IRI activity (compounds **21**-**23**). This activity was dependent upon the length of the glycopeptide, and the compound with five tripeptide repeats (**23**) was found to be the most active at a lower concentration (12.5 *μ*M) in comparison to the compound with three tripeptide repeats (**21**) which was active at a much higher concentration (0.8 mM). AFGP analogues **21**-**23** were found to induce hexagonal ice crystal shaping, suggesting that they are interacting with the ice lattice, however the TH

**Figure 13.** Structures of *C*-linked AFGP analogues containing different side chain lengths between the carbohydrate moiety and the polypeptide backbone, reported by the Ben laboratory. [57,178]

activity of these compounds was not assessed. Irregular incorporation of proline into these derivatives was detrimental to IRI activity as analogues **24**-**26** were only slightly active at a much higher concentration than the alanine-containing derivatives. However, incorporation of proline into a glycopeptide possessing four tripeptide repeats of (Pro-Ala-Thr(GalN‐ HAc))*n* (**27**) resulted in similar IRI activity as the analogue containing four tripeptide repeats of (Ala-Ala-Thr(GalNHAc))*n* (**22**).

Three studies have been reported where AFGP analogues containing triazole rings have been synthesized and assessed for their ability to inhibit ice recrystallization. The triazole ring was incorporated to provide a convergent synthetic approach to these analogues and to overcome the low yields often associated with glycosylation. The key step in the synthesis of these analogues was the Cu(I)-catalyzed Huisgen azide-alkyne cycloaddition (or "click" chemistry). [180-182] In 2009, the Brimble group described the synthesis of two AFGP derivatives in which a furanose carbohydrate moiety was conjugated to a polypeptide backbone with a triazolelinker (Figure 15, compounds **28** and **29**). [61,180] The IRI activity of these derivatives was not assessed, however neither compound exhibited thermal hysteresis or induced dynamic ice shaping. [61] Sewald and co-workers have also reported the synthesis of a number of triazolecontaining AFGP peptoid analogues, three of which were assessed for IRI activity (**30**-**32**, Figure 15), but these analogues failed to inhibit ice recrystallization. [181] Finally, in 2011 the Ben laboratory reported the synthesis of *C*-linked triazole-containing AFGP derivatives **33**-**36** (Figure 15) that were structurally similar to one of their more IRI active glycopeptides reported previously (analogue **12**, Figure 11). [182] While analogues **33**-**36** only exhibited weak IRI activity, this study highlighted the importance of the amide-bond present in the side chain of **12** (Figure 11) and identified this structural feature as crucial for potent IRI activity. Collec‐ tively, the result from these three studies suggest that while utilizing "click" chemistry to conjugate the carbohydrate moiety to a polypeptide backbone may offer advantages synthet‐ ically, the triazole-linker is detrimental for IRI activity.

**6.2. Synthetic polymers as ice recrystallization inhibitors**

[181] and Ben (**33**-**36**) laboratories. [182]

**Figure 14.** Structures of AFGP analogues reported by Sewald and co-workers. [179]

All of the compounds discussed thus far that have exhibited the ability to inhibit ice recrys‐ tallization have been peptide or glycopeptide-based molecules. While some of these deriva‐

**Figure 15.** Structures of triazole-containing AFGP analogues reported by the Brimble (**28**-**29**), [180] Sewald (**30**-**32**)

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Ice Recrystallization Inhibitors: From Biological Antifreezes to Small Molecules http://dx.doi.org/10.5772/54992 195

**Figure 14.** Structures of AFGP analogues reported by Sewald and co-workers. [179]

activity of these compounds was not assessed. Irregular incorporation of proline into these derivatives was detrimental to IRI activity as analogues **24**-**26** were only slightly active at a much higher concentration than the alanine-containing derivatives. However, incorporation of proline into a glycopeptide possessing four tripeptide repeats of (Pro-Ala-Thr(GalN‐ HAc))*n* (**27**) resulted in similar IRI activity as the analogue containing four tripeptide repeats

**Figure 13.** Structures of *C*-linked AFGP analogues containing different side chain lengths between the carbohydrate

moiety and the polypeptide backbone, reported by the Ben laboratory. [57,178]

Three studies have been reported where AFGP analogues containing triazole rings have been synthesized and assessed for their ability to inhibit ice recrystallization. The triazole ring was incorporated to provide a convergent synthetic approach to these analogues and to overcome the low yields often associated with glycosylation. The key step in the synthesis of these analogues was the Cu(I)-catalyzed Huisgen azide-alkyne cycloaddition (or "click" chemistry). [180-182] In 2009, the Brimble group described the synthesis of two AFGP derivatives in which a furanose carbohydrate moiety was conjugated to a polypeptide backbone with a triazolelinker (Figure 15, compounds **28** and **29**). [61,180] The IRI activity of these derivatives was not assessed, however neither compound exhibited thermal hysteresis or induced dynamic ice shaping. [61] Sewald and co-workers have also reported the synthesis of a number of triazolecontaining AFGP peptoid analogues, three of which were assessed for IRI activity (**30**-**32**, Figure 15), but these analogues failed to inhibit ice recrystallization. [181] Finally, in 2011 the Ben laboratory reported the synthesis of *C*-linked triazole-containing AFGP derivatives **33**-**36** (Figure 15) that were structurally similar to one of their more IRI active glycopeptides reported previously (analogue **12**, Figure 11). [182] While analogues **33**-**36** only exhibited weak IRI activity, this study highlighted the importance of the amide-bond present in the side chain of **12** (Figure 11) and identified this structural feature as crucial for potent IRI activity. Collec‐ tively, the result from these three studies suggest that while utilizing "click" chemistry to conjugate the carbohydrate moiety to a polypeptide backbone may offer advantages synthet‐

of (Ala-Ala-Thr(GalNHAc))*n* (**22**).

194 Recent Developments in the Study of Recrystallization

ically, the triazole-linker is detrimental for IRI activity.

**Figure 15.** Structures of triazole-containing AFGP analogues reported by the Brimble (**28**-**29**), [180] Sewald (**30**-**32**) [181] and Ben (**33**-**36**) laboratories. [182]

#### **6.2. Synthetic polymers as ice recrystallization inhibitors**

All of the compounds discussed thus far that have exhibited the ability to inhibit ice recrys‐ tallization have been peptide or glycopeptide-based molecules. While some of these deriva‐ tives show great promise for the many applications of ice recrystallization inhibitors, the main limitation is that large-scale preparation of these compounds for *in vitro* or *in vivo* applications is problematic. Thus, interest has arisen in small molecules (section 6.3) and synthetic polymers (described below) that can inhibit ice recrystallization. Such compounds can be more efficiently synthesized. Knight *et al.* in 1995 made the first observation that synthetic polymers could inhibit ice recrystallization. [41] In this study it was found that poly-L-histidine, poly-Lhydroxyproline and polyvinyl alcohol (PVA) exhibited IRI activity at concentrations less than 1 mg/mL in pure water, whereas poly-L-aspartic acid, poly-L-asparagine, polyacrylic acid and polyvinylpyrrolidone were inactive. With the exception of PVA, which retained its IRI activity in a NaCl solution, these polypeptides and polymers were not assessed for IRI activity in a salt solution to negate false positive effects. [41] Following this study, the activity of PVA has been further investigated and various synthetic polymers have been examined for their ability to inhibit ice recrystallization.

recrystallization. However, incorporating a different carbohydrate residue (**44**) failed to

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197

The Ben laboratory was the first group to report that small molecules, which were not peptide or polymer-based, could inhibit ice recrystallization. In 2008, Tam *et al.* reported a study examining the correlation between carbohydrate hydration and ice recrystallization inhibition. [177] This study arose from the observation that having a more hydrated carbohydrate moiety on one of their most active *C*-linked AFGP analogues (**12**) was a contributing factor to its exhibited IRI activity (see section 6.1, Figure 12). [58] Consequently, four monosaccharides and five disaccharides with known hydration parameters [174-176] were assessed for their ability to inhibit ice recrystallization. The structures of the mono- and disaccharides along with corresponding hydration numbers, isentropic molar compressibility and partial molar volume values are shown in Table 1. At a concentration of 22 mM, D- galactose exhibited moderate IRI activity, D- glucose and D- mannose had weak activity while D- talose was inactive. [177] These results showed a strong linear correlation between the hydration number of the monosac‐ charides and their respective IRI activity. The disaccharides examined also showed this strong linear correlation of their hydration number to IRI activity. Melibiose exhibited moderate IRI activity, while lactose and trehalose showed weak activity and maltose and sucrose were

The hydration layer or hydration shell of a carbohydrate can be defined as the number of tightly bound water molecules that surround the carbohydrate in aqueous solution. The hydration of carbohydrates has been the focus of many studies, and hypotheses for rationalizing observed hydration characteristics include hydration numbers, [185-188] anomeric effect, [189] hydro‐ philic volume, [190] hydrophobic index, [191] the ratio of axial versus equatorial hydroxyl groups [192,193] and the compatibility with bulk-water based upon the position of the nextnearest-neighbor hydroxyl group. [194,195] In the early 1990s, Galema *et al.* studied key parameters thought to dictate hydration characteristics and these were correlated to carbohy‐ drate stereochemistry. Using kinetic experiments and density ultrasound measurements, the partial molar volumes, isentropic partial molar compressibilities and hydration numbers were determined for many commercially available mono- and disaccharides. [174-176] The isen‐ tropic partial molar compressibility and partial molar volume values of the carbohydrates quantify their "compatibility" with the three-dimensional hydrogen-bond network of bulk-

**Figure 16.** Structures of synthetic polymers assessed for IRI activity. [41,59,95,145]

**6.3. Small molecules as ice recrystallization inhibitors**

increase IRI activity. [59,95]

inactive.

In 2003 Inada *et al.* reported an extensive study on the IRI activity of PVA. The activity of PVA was found to be dependent on its molecular mass, with an increase in activity observed with higher molecular weight polymers of PVA. [145] Polymers with an average molecular weight of ~90 000 g/mol were found to exhibit comparable activity to a type I AFP from winter flounder at similar concentrations. However, due to the large difference in molecular weights between PVA and the AFP, the quantity of PVA required to exhibit this activity was significantly higher than that of the AFP. In 2009, Gibson *et al.* re-examined the molecular weight dependence of PVA and showed that PVA with an average molecular weight of ~115 500 has potent IRI activity at a concentration of 5 mg/mL. [59] It was suggested that the ability of PVA to inhibit ice recrystallization is attributed to its ability to interact with the ice crystal lattice. Budke and Koop reported that PVA induces dynamic ice shaping capabilities and suggested this is occurring as the spacing of the PVA hydroxyl groups are closely matched to that of the prism planes of ice, allowing adsorption to these planes. [183] Furthermore, Inada and Lu have shown that PVA exhibits a small TH gap of 0.037 °C at 50 mg/mL, suggesting that an adsorption to ice is occurring. [157]

In addition to PVA, a number of other water-soluble polymers have also been investigated for their ability to inhibit ice recrystallization. [95] In 2009, Gibson *et al.* reported the IRI activity of various structurally diverse polymers (Figure 16). [59] Polyacrylic acid (PAA, **37**), poly(2 aminoethyl methacrylate) (**38**), polyethylene glycol (PEG, **39**), poly-L-Lysine (**40**) and poly-Lglutamic acid (**41**) exhibited only weak IRI activity, and an increase in concentration did not improve activity for any of these polymers. Poly-L-hydroxyproline (**42**) was found to exhibit IRI activity and this activity was dependent on polymer concentration. Poly-L-hydroxyproline has a PPII helical secondary structure [184] similar to the structure AFGPs are suggested to adopt. However, it was suggested this secondary structure is not required for IRI activity as PVA and poly-L-hydroxyproline exhibited similar IRI activities, but PVA is largely unstruc‐ tured in solution. [59] Two vinyl-derived glycopolymers were also assessed for their ability to inhibit ice recrystallization (**43** and **44**, Figure 16). The highest molecular weight glycopolymer with a glucose residue (**43**, at ~105000 g/mol) did exhibit a moderate ability to inhibit ice recrystallization. However, incorporating a different carbohydrate residue (**44**) failed to increase IRI activity. [59,95]

**Figure 16.** Structures of synthetic polymers assessed for IRI activity. [41,59,95,145]

#### **6.3. Small molecules as ice recrystallization inhibitors**

tives show great promise for the many applications of ice recrystallization inhibitors, the main limitation is that large-scale preparation of these compounds for *in vitro* or *in vivo* applications is problematic. Thus, interest has arisen in small molecules (section 6.3) and synthetic polymers (described below) that can inhibit ice recrystallization. Such compounds can be more efficiently synthesized. Knight *et al.* in 1995 made the first observation that synthetic polymers could inhibit ice recrystallization. [41] In this study it was found that poly-L-histidine, poly-Lhydroxyproline and polyvinyl alcohol (PVA) exhibited IRI activity at concentrations less than 1 mg/mL in pure water, whereas poly-L-aspartic acid, poly-L-asparagine, polyacrylic acid and polyvinylpyrrolidone were inactive. With the exception of PVA, which retained its IRI activity in a NaCl solution, these polypeptides and polymers were not assessed for IRI activity in a salt solution to negate false positive effects. [41] Following this study, the activity of PVA has been further investigated and various synthetic polymers have been examined for their ability to

In 2003 Inada *et al.* reported an extensive study on the IRI activity of PVA. The activity of PVA was found to be dependent on its molecular mass, with an increase in activity observed with higher molecular weight polymers of PVA. [145] Polymers with an average molecular weight of ~90 000 g/mol were found to exhibit comparable activity to a type I AFP from winter flounder at similar concentrations. However, due to the large difference in molecular weights between PVA and the AFP, the quantity of PVA required to exhibit this activity was significantly higher than that of the AFP. In 2009, Gibson *et al.* re-examined the molecular weight dependence of PVA and showed that PVA with an average molecular weight of ~115 500 has potent IRI activity at a concentration of 5 mg/mL. [59] It was suggested that the ability of PVA to inhibit ice recrystallization is attributed to its ability to interact with the ice crystal lattice. Budke and Koop reported that PVA induces dynamic ice shaping capabilities and suggested this is occurring as the spacing of the PVA hydroxyl groups are closely matched to that of the prism planes of ice, allowing adsorption to these planes. [183] Furthermore, Inada and Lu have shown that PVA exhibits a small TH gap of 0.037 °C at 50 mg/mL, suggesting that an adsorption to

In addition to PVA, a number of other water-soluble polymers have also been investigated for their ability to inhibit ice recrystallization. [95] In 2009, Gibson *et al.* reported the IRI activity of various structurally diverse polymers (Figure 16). [59] Polyacrylic acid (PAA, **37**), poly(2 aminoethyl methacrylate) (**38**), polyethylene glycol (PEG, **39**), poly-L-Lysine (**40**) and poly-Lglutamic acid (**41**) exhibited only weak IRI activity, and an increase in concentration did not improve activity for any of these polymers. Poly-L-hydroxyproline (**42**) was found to exhibit IRI activity and this activity was dependent on polymer concentration. Poly-L-hydroxyproline has a PPII helical secondary structure [184] similar to the structure AFGPs are suggested to adopt. However, it was suggested this secondary structure is not required for IRI activity as PVA and poly-L-hydroxyproline exhibited similar IRI activities, but PVA is largely unstruc‐ tured in solution. [59] Two vinyl-derived glycopolymers were also assessed for their ability to inhibit ice recrystallization (**43** and **44**, Figure 16). The highest molecular weight glycopolymer with a glucose residue (**43**, at ~105000 g/mol) did exhibit a moderate ability to inhibit ice

inhibit ice recrystallization.

196 Recent Developments in the Study of Recrystallization

ice is occurring. [157]

The Ben laboratory was the first group to report that small molecules, which were not peptide or polymer-based, could inhibit ice recrystallization. In 2008, Tam *et al.* reported a study examining the correlation between carbohydrate hydration and ice recrystallization inhibition. [177] This study arose from the observation that having a more hydrated carbohydrate moiety on one of their most active *C*-linked AFGP analogues (**12**) was a contributing factor to its exhibited IRI activity (see section 6.1, Figure 12). [58] Consequently, four monosaccharides and five disaccharides with known hydration parameters [174-176] were assessed for their ability to inhibit ice recrystallization. The structures of the mono- and disaccharides along with corresponding hydration numbers, isentropic molar compressibility and partial molar volume values are shown in Table 1. At a concentration of 22 mM, D- galactose exhibited moderate IRI activity, D- glucose and D- mannose had weak activity while D- talose was inactive. [177] These results showed a strong linear correlation between the hydration number of the monosac‐ charides and their respective IRI activity. The disaccharides examined also showed this strong linear correlation of their hydration number to IRI activity. Melibiose exhibited moderate IRI activity, while lactose and trehalose showed weak activity and maltose and sucrose were inactive.

The hydration layer or hydration shell of a carbohydrate can be defined as the number of tightly bound water molecules that surround the carbohydrate in aqueous solution. The hydration of carbohydrates has been the focus of many studies, and hypotheses for rationalizing observed hydration characteristics include hydration numbers, [185-188] anomeric effect, [189] hydro‐ philic volume, [190] hydrophobic index, [191] the ratio of axial versus equatorial hydroxyl groups [192,193] and the compatibility with bulk-water based upon the position of the nextnearest-neighbor hydroxyl group. [194,195] In the early 1990s, Galema *et al.* studied key parameters thought to dictate hydration characteristics and these were correlated to carbohy‐ drate stereochemistry. Using kinetic experiments and density ultrasound measurements, the partial molar volumes, isentropic partial molar compressibilities and hydration numbers were determined for many commercially available mono- and disaccharides. [174-176] The isen‐ tropic partial molar compressibility and partial molar volume values of the carbohydrates quantify their "compatibility" with the three-dimensional hydrogen-bond network of bulk-


talose was the most compatible and caused the least disturbance on the hydrogen-bonded network of bulk-water, whereas D- galactose was the least compatible and caused a greater disturbance on the hydrogen-bonded network of bulk-water. The carbohydrates with an equatorial C4 hydroxyl and either an equatorial or axial C2 hydroxyl group (ie. D- glucose and D- mannose) had a moderate fit and caused a moderate disturbance of the three-dimensional

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199

In the study conducted by Tam *et. al* which investigated the IRI activity of several mono- and disaccharides, a correlation was observed between IRI activity and carbohydrate hydration. [177] As none of the carbohydrates exhibited thermal hysteresis or dynamic ice shaping, it was unlikely that the IRI activity exhibited by the carbohydrates was due to an interaction with the ice lattice. This lead to an alternative proposed mechanism for the inhibition of ice recrystal‐ lization based upon the compatibility of a solute with bulk-water. As described in detail in section 2.0 of this chapter, a semi-ordered quasi-liquid layer (QLL) exists between the highly ordered ice lattice and bulk-water. For ice recrystallization to occur, bulk-water molecules transfer to the QLL, then subsequently from the QLL to the growing ice lattice. [38,39] Tam *et al.* have suggested that the carbohydrates are concentrated at the bulk-water-QLL interface. [177] A carbohydrate that had a poor fit into bulk-water will cause a greater disturbance to its three-dimensional hydrogen-bonded network, increasing the energy associated with the transfer of bulk-water to the QLL. It was therefore hypothesized that the inhibition of ice recrystallization observed with carbohydrates occurred at the bulk-water-QLL interface as more highly hydrated carbohydrates, such as D- galactose, disrupted the pre-ordering of bulkwater making it energetically unfavorable for water molecules to transfer to the QLL. Less hydrated carbohydrates, such as D- talose, fit well into bulk-water and caused less of a disturbance to the pre-ordering of bulk-water, thus inhibition of ice recrystallization was not

The disaccharides assessed in this study also showed a strong linear correlation of their IRI activity to their hydration numbers (values give in table 1). [177] However, the increase in hydration numbers for disaccharides relative to monosaccharides was not reflected with an increase in IRI activity. For instance, melibiose has a hydration number of 15.5, yet it exhibited similar IRI activity to D- galactose, which has a hydration number of 8.7. Furthermore, Dgalactose was significantly more IRI active than maltose, despite maltose having a much larger hydration number (8.7 for D- galactose and 14.5 for maltose). This was attributed to a difference in total steric volume between the monosaccharides (containing one carbohydrate unit) and disaccharides (containing two carbohydrate units). By dividing the carbohydrate hydration number by their partial molar volumes an indication of the degree of hydration per molar volume of carbohydrate was obtained. This value was referred to as the hydration index (HI) and it provided the degree of hydration of the substrate as a function of its size or volume. This metric was useful in justifying why highly hydrated monosaccharides exhibited similar IRI activity as highly hydrated disaccharides at 22 mM, despite hydration numbers for monosaccharides being almost half the value of disaccharides. [177] However, at higher carbohydrate concentrations, such as 220 mM, the disaccharides were twice as IRI active as the monosaccharides. [196] Thus, hydration numbers, not hydration indices, were better

hydrogen-bonded network of bulk-water.

observed.

**Table 1.** Isentropic molar compressibilities (104K2 o(s), cm3 mol-1 bar-1) and hydration numbers of various monosaccharides and disaccharides. Errors of molar compressibility values and hydration numbers are shown in parentheses. [175,177]

water as they are related to the size or volume the carbohydrate occupies upon hydration by water. Hydration numbers are calculated using isentropic coefficients of compressibility and they predict the number of water molecules that are hydrogen-bonded to the carbohydrate. In this study, it was observed that the compatibility of the carbohydrate with the three-dimen‐ sional hydrogen-bond network of bulk-water was directly related to the stereochemical relationship of the hydroxyl groups on the carbohydrate. D- Talose, with axial hydroxyl groups on C2 and C4, had a higher isentropic molar compressibility value and a lower hydration number, and fit well into the three-dimensional hydrogen-bonded network of bulk-water. In contrast, D- galactose, with an axial hydroxyl group on C4 and equatorial hydroxyl group on C2, had a lower isentropic molar compressibility value and a higher hydration number, and had a poor fit into the three-dimensional hydrogen-bonded network of bulk-water. Thus, D- talose was the most compatible and caused the least disturbance on the hydrogen-bonded network of bulk-water, whereas D- galactose was the least compatible and caused a greater disturbance on the hydrogen-bonded network of bulk-water. The carbohydrates with an equatorial C4 hydroxyl and either an equatorial or axial C2 hydroxyl group (ie. D- glucose and D- mannose) had a moderate fit and caused a moderate disturbance of the three-dimensional hydrogen-bonded network of bulk-water.

In the study conducted by Tam *et. al* which investigated the IRI activity of several mono- and disaccharides, a correlation was observed between IRI activity and carbohydrate hydration. [177] As none of the carbohydrates exhibited thermal hysteresis or dynamic ice shaping, it was unlikely that the IRI activity exhibited by the carbohydrates was due to an interaction with the ice lattice. This lead to an alternative proposed mechanism for the inhibition of ice recrystal‐ lization based upon the compatibility of a solute with bulk-water. As described in detail in section 2.0 of this chapter, a semi-ordered quasi-liquid layer (QLL) exists between the highly ordered ice lattice and bulk-water. For ice recrystallization to occur, bulk-water molecules transfer to the QLL, then subsequently from the QLL to the growing ice lattice. [38,39] Tam *et al.* have suggested that the carbohydrates are concentrated at the bulk-water-QLL interface. [177] A carbohydrate that had a poor fit into bulk-water will cause a greater disturbance to its three-dimensional hydrogen-bonded network, increasing the energy associated with the transfer of bulk-water to the QLL. It was therefore hypothesized that the inhibition of ice recrystallization observed with carbohydrates occurred at the bulk-water-QLL interface as more highly hydrated carbohydrates, such as D- galactose, disrupted the pre-ordering of bulkwater making it energetically unfavorable for water molecules to transfer to the QLL. Less hydrated carbohydrates, such as D- talose, fit well into bulk-water and caused less of a disturbance to the pre-ordering of bulk-water, thus inhibition of ice recrystallization was not observed.

The disaccharides assessed in this study also showed a strong linear correlation of their IRI activity to their hydration numbers (values give in table 1). [177] However, the increase in hydration numbers for disaccharides relative to monosaccharides was not reflected with an increase in IRI activity. For instance, melibiose has a hydration number of 15.5, yet it exhibited similar IRI activity to D- galactose, which has a hydration number of 8.7. Furthermore, Dgalactose was significantly more IRI active than maltose, despite maltose having a much larger hydration number (8.7 for D- galactose and 14.5 for maltose). This was attributed to a difference in total steric volume between the monosaccharides (containing one carbohydrate unit) and disaccharides (containing two carbohydrate units). By dividing the carbohydrate hydration number by their partial molar volumes an indication of the degree of hydration per molar volume of carbohydrate was obtained. This value was referred to as the hydration index (HI) and it provided the degree of hydration of the substrate as a function of its size or volume. This metric was useful in justifying why highly hydrated monosaccharides exhibited similar IRI activity as highly hydrated disaccharides at 22 mM, despite hydration numbers for monosaccharides being almost half the value of disaccharides. [177] However, at higher carbohydrate concentrations, such as 220 mM, the disaccharides were twice as IRI active as the monosaccharides. [196] Thus, hydration numbers, not hydration indices, were better

water as they are related to the size or volume the carbohydrate occupies upon hydration by water. Hydration numbers are calculated using isentropic coefficients of compressibility and they predict the number of water molecules that are hydrogen-bonded to the carbohydrate. In this study, it was observed that the compatibility of the carbohydrate with the three-dimen‐ sional hydrogen-bond network of bulk-water was directly related to the stereochemical relationship of the hydroxyl groups on the carbohydrate. D- Talose, with axial hydroxyl groups on C2 and C4, had a higher isentropic molar compressibility value and a lower hydration number, and fit well into the three-dimensional hydrogen-bonded network of bulk-water. In contrast, D- galactose, with an axial hydroxyl group on C4 and equatorial hydroxyl group on C2, had a lower isentropic molar compressibility value and a higher hydration number, and had a poor fit into the three-dimensional hydrogen-bonded network of bulk-water. Thus, D-

monosaccharides and disaccharides. Errors of molar compressibility values and hydration numbers are shown in

o(s), cm3 mol-1 bar-1) and hydration numbers of various

**Table 1.** Isentropic molar compressibilities (104K2

198 Recent Developments in the Study of Recrystallization

parentheses. [175,177]

predictors of IRI activity at this concentration, but ultimately IRI activity still correlated with carbohydrate hydration.

activity and were less active than the β-(1,4)-linked analogue. These disaccharides were not conjugated to the native polypeptide backbone (Ala-Ala-Thr) found in AFGPs to investigate if the same trend was observed with the glycoconjugates. However, this study highlighted how the structural features necessary for TH and IRI activity may be different as the functional groups which were required for the TH activity of AFGPs (see section 5.3, Figure 8) [166] were

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201

**Figure 18.** Structural disaccharide analogues of the native β-D- galactosyl-(1-3)-*N*-acetyl-D- galactosamine disacchar‐

While the small molecules described above had the ability to inhibit ice recrystallization, all exhibited only weak to moderate activity at much higher concentrations than those of the potently IRI active glycoconjugates. However, in 2012 the Ben laboratory reported the first examples of small carbohydrate-based molecules that were extremely potent inhibitors ice recrystallization, some that were highly IRI active at concentrations much lower than 22 mM. To date, these are the most potent IRI active small molecules. The molecules investigated were carbohydrate-based surfactants and hydrogelators (structures shown in Figures 19), two of which were found to exhibit potent IRI activity. [62] The carbohydrate-based non-ionic surfactant β-octyl-D- galactopyranoside (**62**) was highly IRI active, with potent activity reported at 11 mM. In contrast, carbohydrate-based non-ionic surfactant β-octyl-D- glucopyr‐ anoside (**63**) was only weakly active even at 44 mM. These results were in agreement with previous studies were that derivatives of the more highly hydrated D- galactose were signifi‐ cantly better inhibitors of ice recrystallization than derivatives of the less hydrated D- glucose. [58,177] While these carbohydrate-based surfactants were known to form micelles in solution, it was concluded that micelle formation was unrelated to IRI activity. β-octyl-D- galactopyra‐ noside (**62**) was highly active at a concentration well below its critical micelle concentration (CMC) of 30 mM, where as β-octyl-D- glucopyranoside (**63**) did not exhibit an ability to inhibit ice recrystallization even well above its CMC value of 22 mM. [62] Furthermore, other structurally different non-ionic and anionic surfactants exhibited weak to moderate activity at concentrations well above their respective CMC values. None of the non-ionic carbohydratebased surfactants assessed in this study possessed TH activity or dynamic ice shaping abilities, suggesting that the activity exhibited by these compounds was not due to an interaction with

not required for the IRI activity of the disaccharide analogues. [197]

ide found in AFGPs. [197]

the ice lattice.

Following the report that simple commercially available carbohydrates exhibit moderate IRI activity, the Ben laboratory has reported the ability of various other carbohydrate derivatives to inhibit ice recrystallization. Most of these compounds have been derivatives of D- galactose. [177] *C*-allylated derivatives of galactose (**45** and **49**), glucose (**46** and **50**), mannose (**47**) and talose (**48**) were assessed for ice recrystallization inhibition activity (Figure 17) to investigate the influence of a carbon substituent at the C1 position as their most IRI active AFGP analogues were *C*-linked glycoconjugates (see section 6.1). [177] The α-*C*-allyl-glycosides (**45**-**48**) had similar activities as the native monosaccharide units (ie. D- galactose and α-*C*-allyl-galacto‐ pyranoside exhibited similar IRI activities), and the trend of activity for these *C*-linked derivatives was identical to the trend observed with the corresponding native monosacchar‐ ides (ie. galactose was most active and talose was least active). However, the β-*C*-allylglycosides (**49**-**50**) showed a significant decrease in activity in comparison to the native monosaccharides (D- galactose and D- glucose) and the α-linked derivatives. Other D- galactose derivatives have been assessed for their ability to inhibit ice recrystallization, including compounds **51**-**57** (Figure 17). [197,198] All of these derivatives had weak to poor IRI activity, and were less active than native D- galactose.

**Figure 17.** Structures of D- galactose-based analogues assessed for IRI activity by the Ben laboratory. [197,198]

In addition to monosaccharide derivatives, structural analogues of the disaccharide β-Dgalactosyl-(1-3)-α-*N*-acetyl-D- galactosamine found in native AFGPs were investigated for IRI activity. These include disaccharide **58** (Figure 18), a close analogue of the disaccharide found in native AFGPs, regioisomers of **58** where the terminal β–D- galactosyl unit was linked to the C4 or C6 hydroxyl group of the *N*-acetyl-D- galactosamine moiety (**60** and **61**, respectively), and disaccharide **59**, in which the C2 *N*-acetyl group was replaced with a hydroxyl group. [197] These four disaccharides were assessed for IRI activity at 22 mM, and interestingly the most active disaccharide was not the analogue of the disaccharide found in native AFGPs. The β-(1,4)-linked disaccharide **60** was the most active disaccharide analogue assessed. The β-(1,6) linked disaccharide **61** and both β-(1,3)-linked disaccharides, **58**-**59**, exhibited similar IRI activity and were less active than the β-(1,4)-linked analogue. These disaccharides were not conjugated to the native polypeptide backbone (Ala-Ala-Thr) found in AFGPs to investigate if the same trend was observed with the glycoconjugates. However, this study highlighted how the structural features necessary for TH and IRI activity may be different as the functional groups which were required for the TH activity of AFGPs (see section 5.3, Figure 8) [166] were not required for the IRI activity of the disaccharide analogues. [197]

predictors of IRI activity at this concentration, but ultimately IRI activity still correlated with

Following the report that simple commercially available carbohydrates exhibit moderate IRI activity, the Ben laboratory has reported the ability of various other carbohydrate derivatives to inhibit ice recrystallization. Most of these compounds have been derivatives of D- galactose. [177] *C*-allylated derivatives of galactose (**45** and **49**), glucose (**46** and **50**), mannose (**47**) and talose (**48**) were assessed for ice recrystallization inhibition activity (Figure 17) to investigate the influence of a carbon substituent at the C1 position as their most IRI active AFGP analogues were *C*-linked glycoconjugates (see section 6.1). [177] The α-*C*-allyl-glycosides (**45**-**48**) had similar activities as the native monosaccharide units (ie. D- galactose and α-*C*-allyl-galacto‐ pyranoside exhibited similar IRI activities), and the trend of activity for these *C*-linked derivatives was identical to the trend observed with the corresponding native monosacchar‐ ides (ie. galactose was most active and talose was least active). However, the β-*C*-allylglycosides (**49**-**50**) showed a significant decrease in activity in comparison to the native monosaccharides (D- galactose and D- glucose) and the α-linked derivatives. Other D- galactose derivatives have been assessed for their ability to inhibit ice recrystallization, including compounds **51**-**57** (Figure 17). [197,198] All of these derivatives had weak to poor IRI activity,

**Figure 17.** Structures of D- galactose-based analogues assessed for IRI activity by the Ben laboratory. [197,198]

In addition to monosaccharide derivatives, structural analogues of the disaccharide β-Dgalactosyl-(1-3)-α-*N*-acetyl-D- galactosamine found in native AFGPs were investigated for IRI activity. These include disaccharide **58** (Figure 18), a close analogue of the disaccharide found in native AFGPs, regioisomers of **58** where the terminal β–D- galactosyl unit was linked to the C4 or C6 hydroxyl group of the *N*-acetyl-D- galactosamine moiety (**60** and **61**, respectively), and disaccharide **59**, in which the C2 *N*-acetyl group was replaced with a hydroxyl group. [197] These four disaccharides were assessed for IRI activity at 22 mM, and interestingly the most active disaccharide was not the analogue of the disaccharide found in native AFGPs. The β-(1,4)-linked disaccharide **60** was the most active disaccharide analogue assessed. The β-(1,6) linked disaccharide **61** and both β-(1,3)-linked disaccharides, **58**-**59**, exhibited similar IRI

carbohydrate hydration.

200 Recent Developments in the Study of Recrystallization

and were less active than native D- galactose.

**Figure 18.** Structural disaccharide analogues of the native β-D- galactosyl-(1-3)-*N*-acetyl-D- galactosamine disacchar‐ ide found in AFGPs. [197]

While the small molecules described above had the ability to inhibit ice recrystallization, all exhibited only weak to moderate activity at much higher concentrations than those of the potently IRI active glycoconjugates. However, in 2012 the Ben laboratory reported the first examples of small carbohydrate-based molecules that were extremely potent inhibitors ice recrystallization, some that were highly IRI active at concentrations much lower than 22 mM. To date, these are the most potent IRI active small molecules. The molecules investigated were carbohydrate-based surfactants and hydrogelators (structures shown in Figures 19), two of which were found to exhibit potent IRI activity. [62] The carbohydrate-based non-ionic surfactant β-octyl-D- galactopyranoside (**62**) was highly IRI active, with potent activity reported at 11 mM. In contrast, carbohydrate-based non-ionic surfactant β-octyl-D- glucopyr‐ anoside (**63**) was only weakly active even at 44 mM. These results were in agreement with previous studies were that derivatives of the more highly hydrated D- galactose were signifi‐ cantly better inhibitors of ice recrystallization than derivatives of the less hydrated D- glucose. [58,177] While these carbohydrate-based surfactants were known to form micelles in solution, it was concluded that micelle formation was unrelated to IRI activity. β-octyl-D- galactopyra‐ noside (**62**) was highly active at a concentration well below its critical micelle concentration (CMC) of 30 mM, where as β-octyl-D- glucopyranoside (**63**) did not exhibit an ability to inhibit ice recrystallization even well above its CMC value of 22 mM. [62] Furthermore, other structurally different non-ionic and anionic surfactants exhibited weak to moderate activity at concentrations well above their respective CMC values. None of the non-ionic carbohydratebased surfactants assessed in this study possessed TH activity or dynamic ice shaping abilities, suggesting that the activity exhibited by these compounds was not due to an interaction with the ice lattice.

to ensure cells survive the process. Unfortunately, all cryoprotectants exhibit cytoxicity and this complicates the cryopreservation process as the cryoprotectant must often be removed

Ice Recrystallization Inhibitors: From Biological Antifreezes to Small Molecules

10% dimethyl sulfoxide (DMSO) is sufficient for all cryopreservation applications. Unfortu‐ nately, this is incorrect and there is an urgent need for novel cryoprotectants, especially in light of the recent developments in the field of regenerative medicine where the supply of various progenitor cells is problematic for the many clinical applications. To highlight the complexity of this process and the need for new and improved cryoprotectants a brief description of

Traditionally, there exist three characterized mechanisms of cell death that occur during cryopreservation. These are cell rupture due to damage to the external cell membrane, necrosis and cold induced apoptosis. Cell rupture is usually the result of osmotic imbalance causing a loss in membrane integrity. [50] Cell necrosis is characterized by cellular swelling (due to an increase in immune response), compromised cell membrane integrity, random DNA frag‐ mentation by cellular endonucleases, cell lysis and the release of cytokines. Apoptosis (programmed cell death) is a highly complex and closely regulated biochemical pathway (the details of which will not be covered in this chapter). It may appear at first that cell death due to apoptosis is not related to cryopreservation however, it has been demonstrated that cold-

The formation of ice under typical cryopreservation conditions is inevitable, but cooling rates become extremely important in mitigating the damage associated with ice formation. For every cell type there is an optimal cooling and warming rate that is determined by the permeability of the cell membrane to water and the cryoprotectant. Hence, cryopreservation is performed with either slow or fast cooling rates depending on cell type. In most instances, ice will prefer to form outside of the cell. [200] Formation of extracellular ice creates an increased osmotic pressure across the cell membrane. This "osmotic flux" intensifies as ice growth continues after the nucleation event. As the ice crystal grows all solutes are excluded from the ice lattice [201] and are concentrated in the extracellular medium. Cells with less permeable membranes will

The process of dehydration during freezing is somewhat of a "double-edged sword". In one instance, the amount of intracellular water decreases, reducing the chance for intracellular ice formation – a lethal process. However, it has been shown that dehydration and exposure to excessively high concentrations of electrolytes is also lethal to the cell. [202] This is referred to as solute damage or the "solute effect" and it facilitates damage to the cell membrane that is irreparable. [202] Conversely, when cells are frozen very slowly, dehydration and excessive cell shrinkage facilitates cell death. Excessive dehydration can be prevented using cryopro‐ tectants. Two classes of cryoprotectant are commonly employed. Non-penetrating cryopro‐ tectants do not cross the cell membrane and hence remain outside the cell, thereby increasing the osmolality of the extracellular solution, facilitating dehydration of the cell prior to freezing and preventing formation of intracellular ice. Penetrating cryoprotectants, such as DMSO and

rupture with increasing osmotic pressure if they cannot dehydrate fast enough.

C/min with

203

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during the thawing cycle. Indeed there is a common myth that cooling rates of 1 o

cellular injury during cryopreservation will be presented in the following section.

**7.1. The complex mechanisms of cryoinjury**

induced apoptosis is common in cryopreserved cells. [50,199]

**Figure 19.** Structures of carbohydrate-based non-ionic surfactants and hydrogelators assessed for IRI activity by the Ben laboratory. [62] β-octyl-D- galactopyranoside (**62**) and *N*-octyl-D- gluconamide (**64**) are the first report of potent small molecule ice recrystallization inhibitors.

The second class of compounds investigated were carbohydrate-based hydrogelators, as in aqueous solution they were known to aggregate and sequester bulk-water forming fibres and hydrogels. D- glucose hydrogelator derivative *N*-octyl-D- gluconamide (**64**) was found to be a potent inhibitor of ice recrystallization at 0.5 mM, a concentration much lower than that of other reported carbohydrate derivatives. [62] However, the D- galactose hydrogelator deriv‐ ative *N*-octyl-D- galactonamide (**65**) was only weakly IRI active at this same concentration. *N*octyl-D- gluconamide (**64**) is the first example of a small molecule exhibiting potent activity at a concentration much lower than 22 mM, and it was also the first example of a glucose-based derivative exhibiting better activity than a galactose-based derivative. Structure-function work conducted in this study suggested that the amide bond in **64** is an essential structural feature for its activity as **66**-**68** (Figure 19) were significantly less active at much higher concentrations. While *N*-octyl-D- gluconamide (**64**) was able to form hydrogels in solution, it was concluded using solid-state NMR studies and characterization of the hydrogels that the ability to form a hydrogel was not a prerequisite for potent IRI activity. This conclusion was further supported by the fact that *N*-octyl-D- galactonamide (**65**) also formed hydrogels in solution, yet it did not possess IRI activity. Finally, these studies also suggested that ice binding was not a prerequisite for potent activity as solid-state NMR studies and TH measurements failed to indicate an interaction with the ice lattice. To date, the report that small molecules can exhibit potent IRI activity remains a significant discovery that will facilitate the rational design of small molecule ice recrystallization inhibitors suitable for medical, commercial and industrial applications.

### **7. Cryopreservation**

Cryopreservation is a very attractive process for the preservation of biological materials. While vitrification and hypothermic storage each offer their own unique advantages and their own limitations, cryopreservation has a major advantage. At the temperatures associated with cryopreservation (typically -190 o C) all biochemical processes are effectively stopped. How‐ ever, cryopreservation is a complex process during which careful attention to sample volume, cooling rates and cryoprotectants (dimethyl sulfoxide and glycerol) are extremely important to ensure cells survive the process. Unfortunately, all cryoprotectants exhibit cytoxicity and this complicates the cryopreservation process as the cryoprotectant must often be removed during the thawing cycle. Indeed there is a common myth that cooling rates of 1 o C/min with 10% dimethyl sulfoxide (DMSO) is sufficient for all cryopreservation applications. Unfortu‐ nately, this is incorrect and there is an urgent need for novel cryoprotectants, especially in light of the recent developments in the field of regenerative medicine where the supply of various progenitor cells is problematic for the many clinical applications. To highlight the complexity of this process and the need for new and improved cryoprotectants a brief description of cellular injury during cryopreservation will be presented in the following section.

#### **7.1. The complex mechanisms of cryoinjury**

**Figure 19.** Structures of carbohydrate-based non-ionic surfactants and hydrogelators assessed for IRI activity by the Ben laboratory. [62] β-octyl-D- galactopyranoside (**62**) and *N*-octyl-D- gluconamide (**64**) are the first report of potent

The second class of compounds investigated were carbohydrate-based hydrogelators, as in aqueous solution they were known to aggregate and sequester bulk-water forming fibres and hydrogels. D- glucose hydrogelator derivative *N*-octyl-D- gluconamide (**64**) was found to be a potent inhibitor of ice recrystallization at 0.5 mM, a concentration much lower than that of other reported carbohydrate derivatives. [62] However, the D- galactose hydrogelator deriv‐ ative *N*-octyl-D- galactonamide (**65**) was only weakly IRI active at this same concentration. *N*octyl-D- gluconamide (**64**) is the first example of a small molecule exhibiting potent activity at a concentration much lower than 22 mM, and it was also the first example of a glucose-based derivative exhibiting better activity than a galactose-based derivative. Structure-function work conducted in this study suggested that the amide bond in **64** is an essential structural feature for its activity as **66**-**68** (Figure 19) were significantly less active at much higher concentrations. While *N*-octyl-D- gluconamide (**64**) was able to form hydrogels in solution, it was concluded using solid-state NMR studies and characterization of the hydrogels that the ability to form a hydrogel was not a prerequisite for potent IRI activity. This conclusion was further supported by the fact that *N*-octyl-D- galactonamide (**65**) also formed hydrogels in solution, yet it did not possess IRI activity. Finally, these studies also suggested that ice binding was not a prerequisite for potent activity as solid-state NMR studies and TH measurements failed to indicate an interaction with the ice lattice. To date, the report that small molecules can exhibit potent IRI activity remains a significant discovery that will facilitate the rational design of small molecule ice recrystallization inhibitors suitable for medical, commercial and industrial applications.

Cryopreservation is a very attractive process for the preservation of biological materials. While vitrification and hypothermic storage each offer their own unique advantages and their own limitations, cryopreservation has a major advantage. At the temperatures associated with

ever, cryopreservation is a complex process during which careful attention to sample volume, cooling rates and cryoprotectants (dimethyl sulfoxide and glycerol) are extremely important

C) all biochemical processes are effectively stopped. How‐

small molecule ice recrystallization inhibitors.

202 Recent Developments in the Study of Recrystallization

**7. Cryopreservation**

cryopreservation (typically -190 o

Traditionally, there exist three characterized mechanisms of cell death that occur during cryopreservation. These are cell rupture due to damage to the external cell membrane, necrosis and cold induced apoptosis. Cell rupture is usually the result of osmotic imbalance causing a loss in membrane integrity. [50] Cell necrosis is characterized by cellular swelling (due to an increase in immune response), compromised cell membrane integrity, random DNA frag‐ mentation by cellular endonucleases, cell lysis and the release of cytokines. Apoptosis (programmed cell death) is a highly complex and closely regulated biochemical pathway (the details of which will not be covered in this chapter). It may appear at first that cell death due to apoptosis is not related to cryopreservation however, it has been demonstrated that coldinduced apoptosis is common in cryopreserved cells. [50,199]

The formation of ice under typical cryopreservation conditions is inevitable, but cooling rates become extremely important in mitigating the damage associated with ice formation. For every cell type there is an optimal cooling and warming rate that is determined by the permeability of the cell membrane to water and the cryoprotectant. Hence, cryopreservation is performed with either slow or fast cooling rates depending on cell type. In most instances, ice will prefer to form outside of the cell. [200] Formation of extracellular ice creates an increased osmotic pressure across the cell membrane. This "osmotic flux" intensifies as ice growth continues after the nucleation event. As the ice crystal grows all solutes are excluded from the ice lattice [201] and are concentrated in the extracellular medium. Cells with less permeable membranes will rupture with increasing osmotic pressure if they cannot dehydrate fast enough.

The process of dehydration during freezing is somewhat of a "double-edged sword". In one instance, the amount of intracellular water decreases, reducing the chance for intracellular ice formation – a lethal process. However, it has been shown that dehydration and exposure to excessively high concentrations of electrolytes is also lethal to the cell. [202] This is referred to as solute damage or the "solute effect" and it facilitates damage to the cell membrane that is irreparable. [202] Conversely, when cells are frozen very slowly, dehydration and excessive cell shrinkage facilitates cell death. Excessive dehydration can be prevented using cryopro‐ tectants. Two classes of cryoprotectant are commonly employed. Non-penetrating cryopro‐ tectants do not cross the cell membrane and hence remain outside the cell, thereby increasing the osmolality of the extracellular solution, facilitating dehydration of the cell prior to freezing and preventing formation of intracellular ice. Penetrating cryoprotectants, such as DMSO and glycerol, readily cross the cell membrane and decrease the concentration of intracellular electrolytes while maintaining greater cell volumes. The major problem with penetrating cryoprotectants is cytotoxicity due to the disruption of intracellular signaling. [203] In summary, cryopreservation of cells using slow-freezing results in dehydration of the cell in response to increasing osmotic pressures as electrolytes are concentrated outside the cell during extracellular ice growth. While dehydration of the cells helps to prevent intracellular ice growth, it is also detrimental to cell survival.

ma and bovine immature oocytes. Furthermore, these oolemma and oocytes underwent successful *in vitro* maturation and fertilization. [211,212] In addition, it has been shown that AFPs can stabilize plasma membranes. [213] Crowe and co-workers demonstrated that while a 1 mg/mL solution of AFGP prevented cold-induced activation of human blood platelets following hypothermic storage, a type I AFP had no effect. [214,215] De‐ spite these promising examples, toxic effects during hypothermic storage from the BAs during hypothermic storage have also been reported. Both AFPs and AFGPs have exhib‐ ited significant toxic effects and have compromised cell viabilities in spinach thylakoids,

Ice Recrystallization Inhibitors: From Biological Antifreezes to Small Molecules

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205

In addition to hypotherminc storage, BAs have also been utilized for cryostorage of biological materials. Several studies have reported benefits of using AFPs and AFGPs as cryoprotectants. Rubinsky and co-workers observed dramatically improved morphological integrity of immature oocytes and two-cell-stage embroys of mice and pigs that were subjected to vitrification in the presence of 40 mg/mL AFGPs. [219,220] Similar results were observed with mature mouse oocytes [221], bovine and ovine embryos at the morula/blastocyst stage, [222] ram spermatozoa, [217] chimpanzee spermatozoa [218] and porcine oocytes. [223] While postthaw viabilities were increased in the presence of BAs with ram and chimpanzee spermatozoa and porcine oocytes, cytotoxic effects during cooling were also observed. [217,218,223]

In contrast, other investigations have reported that BAs fail to protect cells during cryopre‐ servation and actually facilitate cellular damage during cryopreservation. For instance, no specific benefits were observed in survival rates of vitrified bovine blastocysts, [224] two-stepcryopreserved oyster oocytes [225] and equine embroys using various AFPs. [226] Freezing of red blood cells in the presence of glycerol with AFPs (at concentrations between 25 and 1000 *μ*g/mL) [227] and AFGPs (at 40 *μ*g/mL) has been reported to damage cells during cryopreser‐ vation. [228] A similar result was also observed during the cryopreservation of hematopoietic cells with AFPs in DMSO. [229] Additionally, this cellular damage during cryopreservation with BAs has also been reported with spinach thylakoids, [216] intact rat heart (from cardiac explant) [230] and cardiomyocytes. [231] This damage has been attributed to the change in ice crystal morphology that is induced in the presence of BAs (dynamic ice shaping). [228,231] Furthermore, it has been suggested that BAs may also increase the incidence of intracellular ice formation, thereby decreasing cell viabilities post-thaw. [232] Finally, reports have demonstrated both beneficial and detrimental effects with BAs during cryopreservations, depending on AFP concentration and type. [233] At low concentrations AFPs were reported to increase the survival rate of red blood cells however, at higher concentrations where the ice recrystallization inhibition ability of the AFP was significantly enhanced, they decreased

In contrast to native biological antifreezes, the benefit of analogues possessing "customtailored" antifreeze activity for cryopreservation has been demonstrated. In 2011, the Ben laboratory demonstrated that *C*-linked AFGP analogues that exhibit potent IRI activity but not TH activity function as effective cryoprotectants. Using a human embryonic liver cell line, 1.0-1.5 mg/mL of *C*-linked AFGP analogues **11** or **12** doubled cell viability relative to the negative control (cell medium only). [236] The post-thaw viability was comparable to that

[216] ram spermatozoa [217] and chimpanzee spermatozoa. [218]

survival rates. [234,235]

Cryopreservation using high cooling rates traps water inside the cell promoting the for‐ mation intracellular ice. [204] The exact mechanism by which this occurs is not clear [205] however, most cryobiologists believe that intracellular ice formation results in cell death. Hence, practical fast-freezing protocols must dehydrate cells prior to freezing in order to mitigate intracellular ice formation. [206] Of course cryoprotectants are necessary to accomplish this, but the role of the cryoprotectant during fast cooling is different than during slow cooling. Non-penetrating cryoprotectants are employed in an effort to dehy‐ drate the cell and minimize the chance of intracellular ice formation. Interestingly, while the correlation between intracellular ice formation and cell death has been recognized, there is evidence to suggest that formation of intracellular ice does not directly kill cells. [200] Studies have shown that survival of cells post-cryopreservation is dependent upon the rate at which the cells are warmed during thawing and that cell death associated with intracellular ice formation is not caused by the initial nucleation of ice but by an al‐ ternate process during warming. [207,208] Possible mechanisms by which intracellular ice damages cells have been reviewed extensively in the literature and it has been concluded that cell death is occurring as a result of ice recrystallization. [202,209] This hypothesis is supported by the fact that may freeze-tolerant organisms inhabiting sub-zero environ‐ ments produce large quantities of recrystallization-inhibitors *in vivo* to ensure survival. [139,210] In addition, mechanical damage to cell membranes from ice recrystallization has been identified as a primary cause of cell injury during cryopreservation. [50]

#### **7.2. Preservation of biological materials using biological antifreezes and their analogues**

Cellular damage due to ice recrystallization occurs during the storage and thawing cycles of cryopreservation and, given the cryoprotective nature of BAs, it is not surprising that they have been investigated as cryoprotectants to increase cell viability post-thaw. In principle, BAs have the advantage of being relatively non-toxic compared to common cryoprotectants such as DMSO and glycerol. While BAs seem like ideal cryoprotectants, they have not been very effective and often fail to protect mammalian cells from cryoinjury at temperatures outside of the TH gap. This section will discuss specific examples where BAs were used to cryopreserve biological materials, including the benefits and problems associated with their use.

BAs have been examined as protective agents for the hypothermic storage and cryopre‐ servation of various biological materials. AFPs have been reported to protect cell mem‐ branes during hypothermic storage. For instance, Rubinsky and co-workers demonstrated that AFPs [211] and AFGPs [212] of various molecular weights and in concentrations ranging from 1-40 mg/mL can successfully preserve the structural integrity of pig oolem‐ ma and bovine immature oocytes. Furthermore, these oolemma and oocytes underwent successful *in vitro* maturation and fertilization. [211,212] In addition, it has been shown that AFPs can stabilize plasma membranes. [213] Crowe and co-workers demonstrated that while a 1 mg/mL solution of AFGP prevented cold-induced activation of human blood platelets following hypothermic storage, a type I AFP had no effect. [214,215] De‐ spite these promising examples, toxic effects during hypothermic storage from the BAs during hypothermic storage have also been reported. Both AFPs and AFGPs have exhib‐ ited significant toxic effects and have compromised cell viabilities in spinach thylakoids, [216] ram spermatozoa [217] and chimpanzee spermatozoa. [218]

glycerol, readily cross the cell membrane and decrease the concentration of intracellular electrolytes while maintaining greater cell volumes. The major problem with penetrating cryoprotectants is cytotoxicity due to the disruption of intracellular signaling. [203] In summary, cryopreservation of cells using slow-freezing results in dehydration of the cell in response to increasing osmotic pressures as electrolytes are concentrated outside the cell during extracellular ice growth. While dehydration of the cells helps to prevent intracellular

Cryopreservation using high cooling rates traps water inside the cell promoting the for‐ mation intracellular ice. [204] The exact mechanism by which this occurs is not clear [205] however, most cryobiologists believe that intracellular ice formation results in cell death. Hence, practical fast-freezing protocols must dehydrate cells prior to freezing in order to mitigate intracellular ice formation. [206] Of course cryoprotectants are necessary to accomplish this, but the role of the cryoprotectant during fast cooling is different than during slow cooling. Non-penetrating cryoprotectants are employed in an effort to dehy‐ drate the cell and minimize the chance of intracellular ice formation. Interestingly, while the correlation between intracellular ice formation and cell death has been recognized, there is evidence to suggest that formation of intracellular ice does not directly kill cells. [200] Studies have shown that survival of cells post-cryopreservation is dependent upon the rate at which the cells are warmed during thawing and that cell death associated with intracellular ice formation is not caused by the initial nucleation of ice but by an al‐ ternate process during warming. [207,208] Possible mechanisms by which intracellular ice damages cells have been reviewed extensively in the literature and it has been concluded that cell death is occurring as a result of ice recrystallization. [202,209] This hypothesis is supported by the fact that may freeze-tolerant organisms inhabiting sub-zero environ‐ ments produce large quantities of recrystallization-inhibitors *in vivo* to ensure survival. [139,210] In addition, mechanical damage to cell membranes from ice recrystallization has

been identified as a primary cause of cell injury during cryopreservation. [50]

biological materials, including the benefits and problems associated with their use.

**7.2. Preservation of biological materials using biological antifreezes and their analogues**

Cellular damage due to ice recrystallization occurs during the storage and thawing cycles of cryopreservation and, given the cryoprotective nature of BAs, it is not surprising that they have been investigated as cryoprotectants to increase cell viability post-thaw. In principle, BAs have the advantage of being relatively non-toxic compared to common cryoprotectants such as DMSO and glycerol. While BAs seem like ideal cryoprotectants, they have not been very effective and often fail to protect mammalian cells from cryoinjury at temperatures outside of the TH gap. This section will discuss specific examples where BAs were used to cryopreserve

BAs have been examined as protective agents for the hypothermic storage and cryopre‐ servation of various biological materials. AFPs have been reported to protect cell mem‐ branes during hypothermic storage. For instance, Rubinsky and co-workers demonstrated that AFPs [211] and AFGPs [212] of various molecular weights and in concentrations ranging from 1-40 mg/mL can successfully preserve the structural integrity of pig oolem‐

ice growth, it is also detrimental to cell survival.

204 Recent Developments in the Study of Recrystallization

In addition to hypotherminc storage, BAs have also been utilized for cryostorage of biological materials. Several studies have reported benefits of using AFPs and AFGPs as cryoprotectants. Rubinsky and co-workers observed dramatically improved morphological integrity of immature oocytes and two-cell-stage embroys of mice and pigs that were subjected to vitrification in the presence of 40 mg/mL AFGPs. [219,220] Similar results were observed with mature mouse oocytes [221], bovine and ovine embryos at the morula/blastocyst stage, [222] ram spermatozoa, [217] chimpanzee spermatozoa [218] and porcine oocytes. [223] While postthaw viabilities were increased in the presence of BAs with ram and chimpanzee spermatozoa and porcine oocytes, cytotoxic effects during cooling were also observed. [217,218,223]

In contrast, other investigations have reported that BAs fail to protect cells during cryopre‐ servation and actually facilitate cellular damage during cryopreservation. For instance, no specific benefits were observed in survival rates of vitrified bovine blastocysts, [224] two-stepcryopreserved oyster oocytes [225] and equine embroys using various AFPs. [226] Freezing of red blood cells in the presence of glycerol with AFPs (at concentrations between 25 and 1000 *μ*g/mL) [227] and AFGPs (at 40 *μ*g/mL) has been reported to damage cells during cryopreser‐ vation. [228] A similar result was also observed during the cryopreservation of hematopoietic cells with AFPs in DMSO. [229] Additionally, this cellular damage during cryopreservation with BAs has also been reported with spinach thylakoids, [216] intact rat heart (from cardiac explant) [230] and cardiomyocytes. [231] This damage has been attributed to the change in ice crystal morphology that is induced in the presence of BAs (dynamic ice shaping). [228,231] Furthermore, it has been suggested that BAs may also increase the incidence of intracellular ice formation, thereby decreasing cell viabilities post-thaw. [232] Finally, reports have demonstrated both beneficial and detrimental effects with BAs during cryopreservations, depending on AFP concentration and type. [233] At low concentrations AFPs were reported to increase the survival rate of red blood cells however, at higher concentrations where the ice recrystallization inhibition ability of the AFP was significantly enhanced, they decreased survival rates. [234,235]

In contrast to native biological antifreezes, the benefit of analogues possessing "customtailored" antifreeze activity for cryopreservation has been demonstrated. In 2011, the Ben laboratory demonstrated that *C*-linked AFGP analogues that exhibit potent IRI activity but not TH activity function as effective cryoprotectants. Using a human embryonic liver cell line, 1.0-1.5 mg/mL of *C*-linked AFGP analogues **11** or **12** doubled cell viability relative to the negative control (cell medium only). [236] The post-thaw viability was comparable to that obtained with a 2.5% DMSO solution. This effect was attributed to the IRI activity of these *C*linked AFGP analogues. This conclusion was validated when it was demonstrated that IRI active carbohydrates exhibiting minimal cytotoxicity significantly increased cell viabilities post-thaw. [196] To date, these are the only examples where potent inhibitors of ice recrystal‐ lization not displaying thermal hysteresis activity or dynamic ice shaping capabilities have been successfully utilized as cryoprotectants.

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207

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