**5.1. Structures of Biological Antifreezes (BAs)**

below -10 °C. [42] However, Ostwald ripening of polycrystalline ice in an aqueous solution considers the whole ice crystal/liquid water system and thus accounts for the presence of bulkwater and the QLL. In ice, Ostwald ripening is the thermodynamically driven process whereby large ice crystals grow larger at the expense of small crystals, resulting in an overall reduction in energy of the ice crystal/bulk-water interface. [44-46] Throughout the Ostwald ripening process a constant ice volume is maintained. Smaller ice crystals have a higher surface area to volume ratio, giving them higher surface free energy since water molecules on the surface are less stable than the water molecules within the ice crystal. [44,45] However, larger ice crystals have a greater volume to surface area ratio and thus are thermodynamically more stable than small ice crystals. As the total overall volume of ice remains constant during the Ostwald ripening process, water molecules transfer from the surface of smaller ice crystals to bulk-water and then are transferred onto the surface of larger ice crystals. The net result is an increase in the average ice crystal size and a decrease in the total number of ice crystals at a constant total

ice volume, resulting in an overall reduction in the free energy of the system. [46]

Ice recrystallization is particularly problematic in the areas of frozen foods and cryopre‐ servation of biological samples (cells, tissues etc.). Freezing of foods is a well-established process as it helps decrease the rates of deterioration. In the last 30 years, the frozen food industry has taken significant steps to improve the freezing and storage process of vari‐ ous food products, recognizing that all frozen food products have a finite shelf. [47] Changes in texture, taste and overall quality of a frozen food product are a direct result of the ice recrystallization process. It is well established that ice morphology is an impor‐ tant factor in determining food texture and quality. For example, ice cream containing

In medicine, cryostorage is an important process to preserve biological materials or precious cell types such as stem cells (or other progenitors) as well as red blood cells. However, as with any cold storage practice, ice recrystallization remains a major problem and is a significant cause of cellular damage and cell death. [49,50] Section 7.1 of this chapter provides a detailed discussion on the role of ice recrystallization in cryo-injury however, to address these problems effective inhibitors of ice recrystallization are urgently required. Naturally occurring biological antifreezes are very effective inhibitors of ice recrystallization. Biological antifreezes (BAs) are peptides or glycopeptides typically found in organisms inhabiting sub-zero environments. The biological purpose of these compounds is to prevent the seeding of ice crystals *in vivo* and

The first biological antifreezes were reported in the late 1950s. [51,52] Given their ability to prevent cryoinjury upon exposure to cold temperatures, [53-55] they have attracted a great

**5. Biological antifreezes as inhibitors of ice recrystallization**

**4. Impact of recrystallization**

182 Recent Developments in the Study of Recrystallization

prevent cryoinjury and death.

small ice crystals has better texture and taste. [48]

In the late 1950s and early 1960s it was observed by Scholander and colleagues that marine teleost fish did not freeze during the winter despite the water temperature being -1.9 °C, over a degree below the freezing point of their blood serum. [51,52] DeVries and Wohlschlag later attributed their survival to the presence of circulating proteins and glycoproteins. [53-55] These proteins later became known as biological antifreezes, specifically antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs). A variety of AFPs and AFGPs have since been identified in a number of different fish, insects, plants and bacteria.

There are four classes of structurally diverse fish AFPs that have been identified. These are type I, [13,63-72] type II, [73-81] type III, [73-77,82-87] and type IV AFPs. [88-90] The four types of fish AFPs have a wide variation in their size, which can range from 3-12 kDa, and in their secondary structures, which can be α-helices, β-rolls, random coils and globular structures. AFGPs are also present in fish, and are comprised of a tripeptide repeat of (Thr-Ala-Ala)*n*, in which the secondary hydroxyl group of threonine is glycosylated with the disaccharide β-Dgalactosyl-(1-3)-α-*N*-acetyl-D- galactosamine (structure shown in Figure 4). [15,55,60,61,91-95] In general, AFGPs have a homologous structure and have been separated into eight subclasses, AFGP 1-8, based on their molecular masses which range from 2.6 kDa (n = 4) to 33.7 kDa (n = 50). [55] Minor sequence variations have been identified in AFGPs where the first alanine residue is replaced by proline, or where the glycosylated threonine residue is occasionally replace by arginine. [96-101] The solution structure of AFGPs has been debated in the literature. Early circular dichroism (CD) and nuclear magnetic resonance (NMR) studies suggested AFGPs adopt an extended random coil structure. [102-107] However, studies have also suggested that they adopt an ordered helix similar to a PPII type II helix. [106,108-110] It has also suggested that they adopt an amphipathic helical structure, with a hydrophilic face containing the exposed hydroxyl groups of the disaccharide moiety and a hydrophobic face containing the exposed methyl groups of the amino acid residues. [72] However, the most recent studies have indicated that AFGP 1-5 possess no form of long-range order and that AFGP-8 is predominantly random coil with short segments of localized order. [106-108] A brief summary of the key structural differences between AFPs and AFGPs is provided in Figure 4.

A number of other AFPs have been identified in other organisms. Various insect AFPs have been identified such as those from the spruce budworm moth (*Choristoneura fumiferana*, *Cf*AFP), [111,112] the yellow mealworm beetle (*Tenebrio molitor*, *Tm*AFP), [113,114] the firecoloured beetle (*Dendroides canadensis*, *Dc*AFP), [115] and the snow flea (sfAFP). [116] Plant AFPs have also been identified from carrot (*Daucus carrota*), [117] bittersweet nightshade (*Solanum dulcamara*), [118] perennial ryegrass (*Lolium perenne*), [119-121] Antarctic hair grass

**Figure 4.** Classification and structural differences between fish antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs).

An illustration of this process is shown in Figure 5B. Inhibiting ice recrystallization results in very small ice crystals within a frozen sample. The ability to maintain small ice crystal size within a frozen solution is a highly desirable property and compounds exhibiting this property

**Figure 5.** Photographs illustrating dynamic ice shaping (DIS) and ice recrystallization inhibition (IRI) activity. **A)** Ice crys‐ tal habit in the presence of 10 mg/mL AFGP-8. The binding of AFGP-8 to the surface of ice crystals induces a change in ice crystal habit, resulting in hexagonal bipyramidal (or spicule) ice crystal shapes. **B)** Photographs of annealed ice grains obtained from a splat-cooling assay. A compound that can inhibit ice recrystallization is able to maintain small

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While there are various methods for assessing IRI activity such as the capillary method assay [140,141] or the use of wide-angle X-ray scattering (WAXS) and differential scanning calorim‐ etry (DSC), [142-144] the most commonly used is the splat-cooling assay. [139] In the splatcooling assay recrystallization can be observed by the change in size of individual ice grains. Briefly, the sample solution is frozen as a thin circular wafer by either dropping a small aliquot onto a precooled (-80 °C) polished aluminum block from a height of approximately 2 meters, [139] or by pressing the solution between two coverslips and freezing. [117] The samples are then annealed at a temperature below 0 °C and the ice crystal size distribution of the sample after a given time is observed. Ice crystal size can be quantified by measuring the mean largest ice grain dimension along any axis [59,145] or by measuring the mean ice grain area. [46,146] Thus, smaller ice crystal sizes represent greater IRI activity. Commonly, analytes are assayed in a salt solution (NaCl, CaCl2 or phosphate buffered saline (PBS)) or a 30-45% sucrose solution, and the solutions without analyte are used as positive controls for ice recrystallization for comparison. The presence of salt or other small solutes is very important as it ensures that liquid is present between ice crystal boundaries and the presence of these solutes negates nonspecific IRI effects that can be observed in pure water. [41] While the original version of this assay was subjective in nature, it has recently been improved using Domain Recognition Software (DRS). [146] IRI can now be reliably quantified, providing accurate comparisons between samples and information on small and subtle changes in IRI activity within a series

**5.3. Biological antifreezes - Mechanisms of action for Thermal Hysteresis (TH) activity**

The most widely accepted mechanism for thermal hysteresis (TH) involves an irreversible adsorption-inhibition process. [133-137] In this mechanism, BAs irreversibly bind to specific

have tremendous medical, commercial and industrial applications.

ice crystal sizes within a frozen solution.

of analogues.

(*Deschampsia antartica*), [122] and several other species. [123,124] Additionally, AFPs have been identified in fungi and bacteria. [125-130] The secondary structures of the various AFPs from plants and insects are also diverse. [131,132] Regardless of where the AFPs are found or their secondary structure, they are all ice-binding proteins that are crucial for the species survival in the harsh cold environments to which they are exposed.

## **5.2. "Antifreeze" activities of biological antifreezes: Thermal Hysteresis (TH) and Ice Recrystallization Inhibition (IRI) activity**

Biological antifreezes exhibit two types of antifreeze activities. The first and the most studied is thermal hysteresis (TH). This is defined as a selective depression of the freezing point of a solution relative to the melting point. [133-135] TH activity is the direct result of the binding of a BA to the surface of a seeded ice crystal. [136,137] The binding of the BA to the surface of ice facilitates a localized freezing point depression and induces a change in the ice crystal habit. This change in ice crystal habit is referred to as dynamic ice shaping (DIS) and is illustrated in Figure 5A. A more detailed description of this process is described in Section 5.3. The standard assay used to measure TH activity is nanolitre osmometry. [138] In this assay, a single ice crystal in an aqueous solution of the biological antifreeze is obtained, and the growth and behavior of the crystal upon increasing/decreasing the temperature can be observed. TH activity is reported as the difference between the observed freezing and melting points in Kelvin or degrees Celsius.

The second type of antifreeze activity exhibited by biological antifreezes is their ability to inhibit ice recrystallization (referred to as ice recrystallization inhibition (IRI) activity). [41,139]

**Figure 5.** Photographs illustrating dynamic ice shaping (DIS) and ice recrystallization inhibition (IRI) activity. **A)** Ice crys‐ tal habit in the presence of 10 mg/mL AFGP-8. The binding of AFGP-8 to the surface of ice crystals induces a change in ice crystal habit, resulting in hexagonal bipyramidal (or spicule) ice crystal shapes. **B)** Photographs of annealed ice grains obtained from a splat-cooling assay. A compound that can inhibit ice recrystallization is able to maintain small ice crystal sizes within a frozen solution.

An illustration of this process is shown in Figure 5B. Inhibiting ice recrystallization results in very small ice crystals within a frozen sample. The ability to maintain small ice crystal size within a frozen solution is a highly desirable property and compounds exhibiting this property have tremendous medical, commercial and industrial applications.

(*Deschampsia antartica*), [122] and several other species. [123,124] Additionally, AFPs have been identified in fungi and bacteria. [125-130] The secondary structures of the various AFPs from plants and insects are also diverse. [131,132] Regardless of where the AFPs are found or their secondary structure, they are all ice-binding proteins that are crucial for the species survival

**Figure 4.** Classification and structural differences between fish antifreeze proteins (AFPs) and antifreeze glycoproteins

**5.2. "Antifreeze" activities of biological antifreezes: Thermal Hysteresis (TH) and Ice**

Biological antifreezes exhibit two types of antifreeze activities. The first and the most studied is thermal hysteresis (TH). This is defined as a selective depression of the freezing point of a solution relative to the melting point. [133-135] TH activity is the direct result of the binding of a BA to the surface of a seeded ice crystal. [136,137] The binding of the BA to the surface of ice facilitates a localized freezing point depression and induces a change in the ice crystal habit. This change in ice crystal habit is referred to as dynamic ice shaping (DIS) and is illustrated in Figure 5A. A more detailed description of this process is described in Section 5.3. The standard assay used to measure TH activity is nanolitre osmometry. [138] In this assay, a single ice crystal in an aqueous solution of the biological antifreeze is obtained, and the growth and behavior of the crystal upon increasing/decreasing the temperature can be observed. TH activity is reported as the difference between the observed freezing and melting points in Kelvin or

The second type of antifreeze activity exhibited by biological antifreezes is their ability to inhibit ice recrystallization (referred to as ice recrystallization inhibition (IRI) activity). [41,139]

in the harsh cold environments to which they are exposed.

**Recrystallization Inhibition (IRI) activity**

184 Recent Developments in the Study of Recrystallization

degrees Celsius.

(AFGPs).

While there are various methods for assessing IRI activity such as the capillary method assay [140,141] or the use of wide-angle X-ray scattering (WAXS) and differential scanning calorim‐ etry (DSC), [142-144] the most commonly used is the splat-cooling assay. [139] In the splatcooling assay recrystallization can be observed by the change in size of individual ice grains. Briefly, the sample solution is frozen as a thin circular wafer by either dropping a small aliquot onto a precooled (-80 °C) polished aluminum block from a height of approximately 2 meters, [139] or by pressing the solution between two coverslips and freezing. [117] The samples are then annealed at a temperature below 0 °C and the ice crystal size distribution of the sample after a given time is observed. Ice crystal size can be quantified by measuring the mean largest ice grain dimension along any axis [59,145] or by measuring the mean ice grain area. [46,146] Thus, smaller ice crystal sizes represent greater IRI activity. Commonly, analytes are assayed in a salt solution (NaCl, CaCl2 or phosphate buffered saline (PBS)) or a 30-45% sucrose solution, and the solutions without analyte are used as positive controls for ice recrystallization for comparison. The presence of salt or other small solutes is very important as it ensures that liquid is present between ice crystal boundaries and the presence of these solutes negates nonspecific IRI effects that can be observed in pure water. [41] While the original version of this assay was subjective in nature, it has recently been improved using Domain Recognition Software (DRS). [146] IRI can now be reliably quantified, providing accurate comparisons between samples and information on small and subtle changes in IRI activity within a series of analogues.

#### **5.3. Biological antifreezes - Mechanisms of action for Thermal Hysteresis (TH) activity**

The most widely accepted mechanism for thermal hysteresis (TH) involves an irreversible adsorption-inhibition process. [133-137] In this mechanism, BAs irreversibly bind to specific planes of a growing ice crystal. Preferential binding occurs on the prism faces of ice, thus inhibiting ice growth along the *a*-axis. [93,147-149] Ice crystal growth continues as the tem‐ perature of the solution is decreased below the hysteresis freezing point, however it occurs along the *c*-axis, giving rise to the characteristic hexagonal bipyramidal (or spicule) crystal shapes (illustrated in Figure 6). [133,150] The faces that BAs bind to can be determined experimentally by ice hemisphere etching. [136] In this experiment, a single ice crystal in a dilute solution of the BA is grown into a hemisphere such that all interfacial orientations are present during growth. As adsorption of the BA to ice is irreversible, the BA is incorporated into the crystal during growth. Sublimation of the ice crystal then results in visibly etched regions on the ice surface where the BA adsorbed and the orientation of these regions can be observed. While it has been determined that BAs adsorb preferentially to the prism planes of a seeded ice crystal, various insect and plant AFPs adsorb to the basal planes, and it is postulated this results in the superior TH activity exhibited by these proteins. [131,132,151]

**Figure 6.** Formation of hexagonal bipyramidal ice crystals by inhibition of growth on the prism faces due to adsorp‐ tion of BAs.

The irreversible binding of a BA to the surface of ice crystals results in a localized freezing point depression. This occurs *via* the Kelvin (or Gibbs-Thomson) Effect. [135] Given that ice growth cannot occur where the BA has adsorbed, growth occurs on the ice surfaces between adjacent BA molecules, resulting in curved ice surfaces (shown in Figure 7). The energetic cost of adding a water molecule (freezing) to this curved surface is high and it becomes unfavorable for more water molecules to add to this surface, thus a localized freezing point depression is observed. This process does not affect the energetics of the melting process, hence only the freezing point is depressed while the melting point remains constant, resulting in a thermal hysteresis gap (Figure 7A). [135,149,152]

proposed by Knight and DeVries. In this model, the adsorbed BA molecules exhibit inhibition

**Figure 7.** Illustrations of thermal hysteresis (TH) activity and the two models of ice growth inhibition. **A)** BAs have the ability to depress the freezing point of ice crystals relative to the melting point, resulting in a thermal hysteresis gap. **B)** Step-pinning model and **C)** mattress model depicting the irreversible adsorption-inhibition mechanism of BAs.

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Both of these models assume an irreversible adsorption of the BA onto the surface of ice. However, there have been reports suggesting that the adsorption is reversible. The main argument in favour of this is that if adsorption were truly irreversible then significant levels of adsorption would be observed in the presence of very low concentrations of BAs, [67] however this has not been definitively observed. Furthermore, a large free energy of adsorp‐ tion of BAs would be expected, but it has been observed that the free energy of adsorption is close to zero. [153] Consequently, alternative mechanisms have been proposed describing ice

by pinning ice growth normal (perpendicular) to the ice surface. [136]

There are two models that described how BAs inhibit ice growth within the thermal hysteretic gap. The first (illustrated in Figure 7B) was proposed by Raymond and DeVries and is known as the step pinning model. In this model, the growth of a step is inhibited by the BA which has pinned ice growth across the ice surface. [133] However, this model assumes that ice crystal growth occurs in steps advancing across the plane that the BA is adsorbed. The second model (illustrated in Figure 7C) is a three-dimensional model known as the mattress model and was Ice Recrystallization Inhibitors: From Biological Antifreezes to Small Molecules http://dx.doi.org/10.5772/54992 187

planes of a growing ice crystal. Preferential binding occurs on the prism faces of ice, thus inhibiting ice growth along the *a*-axis. [93,147-149] Ice crystal growth continues as the tem‐ perature of the solution is decreased below the hysteresis freezing point, however it occurs along the *c*-axis, giving rise to the characteristic hexagonal bipyramidal (or spicule) crystal shapes (illustrated in Figure 6). [133,150] The faces that BAs bind to can be determined experimentally by ice hemisphere etching. [136] In this experiment, a single ice crystal in a dilute solution of the BA is grown into a hemisphere such that all interfacial orientations are present during growth. As adsorption of the BA to ice is irreversible, the BA is incorporated into the crystal during growth. Sublimation of the ice crystal then results in visibly etched regions on the ice surface where the BA adsorbed and the orientation of these regions can be observed. While it has been determined that BAs adsorb preferentially to the prism planes of a seeded ice crystal, various insect and plant AFPs adsorb to the basal planes, and it is postulated this results in the superior TH activity exhibited by these proteins. [131,132,151]

**Figure 6.** Formation of hexagonal bipyramidal ice crystals by inhibition of growth on the prism faces due to adsorp‐

The irreversible binding of a BA to the surface of ice crystals results in a localized freezing point depression. This occurs *via* the Kelvin (or Gibbs-Thomson) Effect. [135] Given that ice growth cannot occur where the BA has adsorbed, growth occurs on the ice surfaces between adjacent BA molecules, resulting in curved ice surfaces (shown in Figure 7). The energetic cost of adding a water molecule (freezing) to this curved surface is high and it becomes unfavorable for more water molecules to add to this surface, thus a localized freezing point depression is observed. This process does not affect the energetics of the melting process, hence only the freezing point is depressed while the melting point remains constant, resulting in a thermal

There are two models that described how BAs inhibit ice growth within the thermal hysteretic gap. The first (illustrated in Figure 7B) was proposed by Raymond and DeVries and is known as the step pinning model. In this model, the growth of a step is inhibited by the BA which has pinned ice growth across the ice surface. [133] However, this model assumes that ice crystal growth occurs in steps advancing across the plane that the BA is adsorbed. The second model (illustrated in Figure 7C) is a three-dimensional model known as the mattress model and was

tion of BAs.

hysteresis gap (Figure 7A). [135,149,152]

186 Recent Developments in the Study of Recrystallization

**Figure 7.** Illustrations of thermal hysteresis (TH) activity and the two models of ice growth inhibition. **A)** BAs have the ability to depress the freezing point of ice crystals relative to the melting point, resulting in a thermal hysteresis gap. **B)** Step-pinning model and **C)** mattress model depicting the irreversible adsorption-inhibition mechanism of BAs.

proposed by Knight and DeVries. In this model, the adsorbed BA molecules exhibit inhibition by pinning ice growth normal (perpendicular) to the ice surface. [136]

Both of these models assume an irreversible adsorption of the BA onto the surface of ice. However, there have been reports suggesting that the adsorption is reversible. The main argument in favour of this is that if adsorption were truly irreversible then significant levels of adsorption would be observed in the presence of very low concentrations of BAs, [67] however this has not been definitively observed. Furthermore, a large free energy of adsorp‐ tion of BAs would be expected, but it has been observed that the free energy of adsorption is close to zero. [153] Consequently, alternative mechanisms have been proposed describing ice growth inhibition of BAs. [153-156] Regardless of these alternate mechanisms, sufficient data exists to suggest an irreversible adsorption-inhibition mechanism, and consequently this model is the generally accepted mechanism by which BAs exhibit TH activity.

desirable property for a compound to exhibit due to the many potential medical and industrial applications. Furthermore, while BAs do possess potent IRI activity, they cannot be used effectively as cryoprotectants. The ice binding ability associated with the TH activity of BAs alters the habit of ice crystals, and since the temperatures employed during cryopreservation are outside of the TH gap, this exacerbates cellular damage. [168-170] However, during the last several years considerable amount of progress has been made in discovering novel ice recrystallization inhibitors, some of which are synthetic analogues of AFGPs, and the work

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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**

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

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

that has been conducted in this area will be the focus of the next section.

**6. Inhibitors of ice recrystallization**

**inhibitors**

NaCl solutions.

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

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 segment. [166,167]

**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 desirable property for a compound to exhibit due to the many potential medical and industrial applications. Furthermore, while BAs do possess potent IRI activity, they cannot be used effectively as cryoprotectants. The ice binding ability associated with the TH activity of BAs alters the habit of ice crystals, and since the temperatures employed during cryopreservation are outside of the TH gap, this exacerbates cellular damage. [168-170] However, during the last several years considerable amount of progress has been made in discovering novel ice recrystallization inhibitors, some of which are synthetic analogues of AFGPs, and the work that has been conducted in this area will be the focus of the next section.
