El DEL en la experiencia cubana Particularidades, retos y perspectivas.

AFPs and AFGPs are highly effective antifreezes at very low concentrations, however producing large quantities of these proteins is challenging, inefficient and costly (initially the proteins are exacted from fish, for example, and then replicated on a larger scale using genetically modified yeast), thus the development of synthetic analogues is extremely desirable. Currently only a handful of synthetic mimics of AF(G)Ps have been discovered and the research on these tends to be rather limited due to their recent discovery, for example, the first paper on graphene oxide as an antifreeze was published in 2017.245

Polyvinyl Alcohol (PVA)

In 2000, the antifreeze activity of polyvinyl alcohol (Figure 6.17), which has similar structural features to AFPs (hydrophobic and hydrophilic sides),139 was first reported as a potential additive for use in ice-slurry cold-storage systems.237 Subsequently, the antifreeze effects of PVA have been thoroughly researched; it works effectively at con- centrations as low as 0.005 mg/ml229, 238 and shows some thermal hysteresis activity,324 but the link between its structure and antifreeze activity is still unknown. It has been demonstrated that PVA does enhance recovery in the cryopreservation of various cells, including human blood cells, and critically, PVA shows no signs of cytotoxicity at con- centrations as high as 20 mg/ml.325–327

Images of ice crystals grown in the presence of PVA clearly show that PVA can bind to the primary and secondary prism planes of ice, see Figures 6.18 e and f, respectively. Budke and co-workers suggested possible mechanisms for the PVA binding to these planes, based upon aligning every third hydroxyl group of PVA (7.56 ˚A) with every second oxygen atom in the ice lattice (7.36 ˚A), see Figures 6.18 i and j.238 Various research groups have shown that when the concentration of PVA is increased, the ice recrystallisation inhibition activity increases too (Figure 6.19).238, 324, 328, 329

Significant research has demonstrated that the PVA chain length (degree of poly- merisation, DP) has a strong influence on the antifreeze activity; PVA with a DP≥ 19 is required for an effective antifreeze (Figure 6.19b) and a higher DP reduces the con- centration required for antifreeze activity.229, 328, 329 _{Vail and co-workers showed that a}

Figure 6.16: The structures of a) AFGP8 and b) AFP III (North Atlantic Pout) from the Protein Data Bank (PDB ID: 1KDF).

Figure 6.17: The structure of polyvinyl alcohol, where n is the number of repeat units.

Figure 6.18: Images of ice crystals grown in pure water (a-c) and 10 mg/ml DP27 PVA (d-f), the primary (g) and secondary prism (h) planes of ice and the suggested mechanism of binding to these planes (i and j, respectively), where solid lines indicate oxygen atoms in the paper plane and dashed lines are 0.92 ˚A behind. e) and f) demonstrate the effects of PVA binding to the primary and secondary prism planes, respectively. Adapted from Budke et al.238

PVA sample with the same DP but a lower dispersity can be significantly less active,330 hence it is also critical to take the polydispersity of the polymer into account.

Hydrophobicity is known to play a crucial role in IRI.332, 333 Considering that acetate groups are more hydrophobic than hydroxyls, Gibson and co-workers investigated the antifreeze activity of copolymers of PVA and poly vinyl acetate;229 less than 20% acetylation was tolerated by the PVA and more than 25% produced an inactive polymer. Since additional hydrophobic groups reduced the IRI activity, the same measurements were conducted with the addition of hydrophilic groups (N-vinyl pyrrolidone), but these

additions could also only be tolerated at low percentages. The results demonstrate

that the antifreeze activity of PVA is not just due to the presence of hydrophobic or hydrophilic groups, but that the structure and orientation of the functional groups as well.

Safranin Chloride

In 2016 Droriet al. discovered that safranin chloride (referred to as “safranin” from here on) inhibited ice growth in a non-colligative manner.239 Safranin is a small dye molecule that aggregates in solution to form supramolecular stacks (see Figure 6.20a). In addition to this aggregation, the methyl groups, amine groups and the chloride ion all play an important role in the inhibition of ice growth.239 Droriet al. showed that safranin binds reversibly to ice crystals, which form hexagonal plates at lower concentrations (0.49 mg/ml) and bipyramidal needles at higher concentrations (9.82 mg/ml).

When safranin aggregates, theπ-πinteractions in the aromatic regions cause the molecules to form stacks with the phenyl groups on alternating sides (see Figure 6.20a). This results in rows of methyl and amine groups along two sides of the supramolecular stacks which seem to mimic the ice-binding sites found in AFPs. Crystal structures show that the gap between each molecule in the stack is 3.7 ˚A and, due to the alternating pattern in the stack, the distance between two amino groups is 7.4 ˚A, a good match with every second oxygen atom in the ice lattice (7.36 ˚A). This suggests that the pre- organisation of safranin molecules allows the amino groups in safranin to form hydrogen bonds along the primary or secondary prism planes of hexagonal ice, creating a crystal- ice interface.

Phenosafranin chloride (Figure 6.20b), which has the same structure as safranin minus the two methyl groups, exhibits no antifreeze activity demonstrating that the methyl groups are critical for the antifreeze activity of safranin. A comparison of the safranin and phenosafranin chloride crystal structures confirms that the latter does not form supramolecular stacks, which seem to be key for antifreeze activity. Next, the im- portance of the chloride ion was investigated through a comparison of safranin chloride and safranin nitrate (Figure 6.20c). Safranin nitrate showed no signs of antifreeze ac- tivity however, once sodium chloride was added to a safranin nitrate solution, antifreeze activity was observed due to the formation of safranin chloride. Again, a comparison of the crystal structures shows that, although some order is retained, the supramolecular stacks are no longer formed in the case of safranin nitrate, most likely due to the change

Figure 6.19: a) Splat images331 compare the IRI activity of 0.05 and 0.5 mg/ml DP27 PVA with pure water by observing the size of the ice crystals over time. Adapted from Budke et al.238 b) The mean largest grain size (MLGS) of PVA of a range of DPs (10, 19, 30, 56, 154, 246 and 351) and concentrations (0 - 1 mg/ml) relative to a phosphate- buffered saline (PBS) control, highlighting that DP<19 is relatively ineffective. Adapted from Congdon et al.229

Figure 6.20: Chemical structures and crystal structures of a) safranin chloride, b) phenosafranin chloride and c) safranin nitrate. The crystal structures were obtained from the Cambridge Structural Database under the identifiers “YAGTOS”, “YAGTEI” and “YAGTIM” respectively.

in anion size.

Zirconium Acetate and Zirconium Acetate Hydroxide

Zirconium acetate and zirconium acetate hydroxide are two metallic salts which have IRI and DIS activity.240–243 Two ice-structuring mechanisms of these salts have been suggested, both involving the salt forming a hydroxy-bridged polymer-like structure with hydroxyl groups on one side and acetate groups on the other. Even though the exact mechanism is unknown, once again it is clear that there are important similarities between these synthetic analogues and the AF(G)Ps found in nature.

Graphene Oxide

Recently, graphene oxide (GO) has shown evidence of suppressing ice growth and re- stricting recrystallisation of ice crystals due to its hexagonal scaffold.245 In solution, GO forms monomolecular sheets with large oxidised regions containing hydroxyl and epoxy groups on the basal plane and carboxyl groups at the periphery of the structure. The repeating hexagonal carbon rings in the GO sheet cause any surface hydroxy groups to follow this honeycomb-like pattern which matches the structure of hexagonal ice ( note the similarities to the IBSs of the antifreezes previously discussed).

Studies have shown that GO acts as an effective non-colligative antifreeze from concentrations of 0.1 - 5.0 mg/ml and that smaller sheets (10 nm) of GO were more effective than larger sheets (500 nm or 5000 nm). Molecular dynamics studies show that GO has a preference for binding to ice crystals rather than the surrounding liquid water and that a combination of hydrogen bonding and hydrophobic interactions are critical for the antifreeze activity displayed by GO.245 Furthermore, the viability of GO as a cryoprotectant was validated; 0.025 mg/ml GO gave similar results to that of 0.1

mg/ml PVA.245 Within the last year, several studies have shown that GO and carbon

nanomaterials actually show evidence of ice nucleation activity which can be tuned by doping the surfaces.231, 334–336 These are the first synthetic materials found to nucleate ice.337

Metallohelices

In 2017, Gibson and co-workers determined that various amphipathic, metallohelices inhibited ice growth at extremely low concentrations (20 µM).244 Since AF(G)Ps and the other synthetic antifreezes discussed so far have a similar amphipathic structure, these results confirm that amphipathicity might be the key for antifreeze behaviour.