Regioregular Alternating Polyampholytes Have Enhanced Biomimetic Ice Recrystallization Activity Compared to Random Copolymers and the Role of Side Chain versus Main Chain Hydrophobicity

Abstract

Antifreeze proteins from polar fish species are potent ice recrystallization inhibitors (IRIs) effectively stopping all ice growth. Additives that have IRI activity have been shown to enhance cellular cryopreservation with potential to improve the distribution of donor cells and tissue. Polyampholytes, polymers with both anionic and cationic side chains, are a rapidly emerging class of polymer cryoprotectants, but their mode of action and the structural features essential for activity are not clear. Here regioregular polyampholytes are synthesized from maleic anhydride copolymers to enable stoichiometric installation of the charged groups, ensuring regioregularity, which is not possible using conventional random copolymerization. A modular synthetic strategy is employed to enable the backbone and side chain hydrophobicity to be varied, with side chain hydrophobicity found to have a profound effect on the IRI activity. The activity of the regioregular polymers was found to be superior to those derived from a standard random copolymerization with statistical incorporation of monomers, demonstrating that sequence composition is crucial to the activity of IRI active polyampholytes.

Mimicking the Ice Recrystallization Activity of Biological Antifreezes. When is a New Polymer “Active”?

“…Reproduced with permission. [79] Copyright 2017, American Chemical Society. a guide in defining materials' relative function, and to ensure that false positives due to simple colligative effects are not misreported in the literature as the field grows.…”

“…Sequential increases in hydrophobicity were introduced by ring opening of the anhydride using alcohols. [79] There was a clear trend that increased hydrophobicity increased the IRI activity. It is crucial to note here that the magnitude of activity is far less than the previous classes (AF(G)Ps/PVA) with some ice growth still occurring at 20 mg mL −1 , hence care must be taken when discussing potency.…”

“…B) Regioregular alternative copolymers derived from maleic anhydride and vinyl acetate. Reproduced with permission . Copyright 2017, American Chemical Society.…”

“…To study this in more detail, Stubbs et al synthesized a panel of regioregular poly(ampholyte)s using maleic anhydride copolymers, which ensure an alternating, rather than random, copolymer structure. Sequential increases in hydrophobicity were introduced by ring opening of the anhydride using alcohols . There was a clear trend that increased hydrophobicity increased the IRI activity.…”

Designing the next generation of cryoprotectants – From proteins to small molecules

“…While the synthesis of C ‐linked AFGPs was markedly easier than native AFGPs, the solid phase synthesis of these analogs was still not amenable to the large‐scale production required for various applications. As a result, other researchers have synthesized various polymers and polyampholytes which have been assessed for IRI properties. The TH and IRI activity of various polymers (Figure ) containing poly‐ l ‐histidine 12 , poly‐ l ‐hydroxyproline 13 , poly‐ l ‐lysine 14 , poly‐ l ‐asparagine 15 , polyacrylic acid 16 , polyvinylpyrrolidone 17 , polyethylene glycol 18 , and polyvinyl alcohol 19 have been reported .…”