High Molecular Weight Polyproline as a Potential Biosourced Ice Growth Inhibitor: Synthesis, Ice Recrystallization Inhibition, and Specific Ice Face Binding

Abstract

Ice-binding proteins (IBPs) from extremophile organisms can modulate ice formation and growth. There are many (bio)technological applications of IBPs, from cryopreservation to mitigating freeze-thaw damage in concrete to frozen food texture modifiers. Extraction or expression of IBPs can be challenging to scale up, and hence polymeric biomimetics have emerged. It is, however, desirable to use biosourced monomers and heteroatom-containing backbones in polymers for in vivo or environmental applications to allow degradation. Here we investigate high molecular weight polyproline as an ice recrystallization inhibitor (IRI). Low molecular weight polyproline is known to be a weak IRI. Its activity is hypothesized to be due to the unique PPI helix it adopts, but it has not been thoroughly investigated. Here an open-to-air aqueous N-carboxyanhydride polymerization is employed to obtain polyproline with molecular weights of up to 50000 g mol^-1. These polymers were found to have IRI activity down to 5 mg mL^-1, unlike a control peptide of polysarcosine, which did not inhibit all ice growth at up to 40 mg mL^-1. The polyprolines exhibited lower critical solution temperature behavior and assembly/aggregation observed at room temperature, which may contribute to its activity. Single ice crystal assays with polyproline led to faceting, consistent with specific ice-face binding. This work shows that non-vinyl-based polymers can be designed to inhibit ice recrystallization and may offer a more sustainable or environmentally acceptable, while synthetically scalable, route to large-scale applications.

Figure 1

(A) Schematic of the synthetic route followed for the synthesis of PPro_n (n = 20, 52, 103, 206, 515). (B) FTIR of Pro-NCA (left) (highlighting asymmetric carbonyl stretch at 1840 and 1760 cm^–1) and PPro₂₀ (right) (highlighting carbonyl stretch of polymer backbone). (C) Normalized SEC-RI distributions of PPro_n (n = 2k, 5k, 10k) in H₂O:ACN (8:2) + 0.1 M NaNO₃. (D) Diffusion coefficients determined by DOSY for PPro_n (n = 2k, 5k, 10k, 20k, 50k) as a function of targeted molecular weight.

Figure 2

Solution properties of polyproline. (A) CD spectra of PPro of varied DP (1 mg mL^–1 in PBS media). (B) Turbidimetry curves of PPro (5 mg mL^–1 in PBS). (C) Intensity-weighted size distributions of PPro obtained from DLS. (D) Representative cryo-TEM image of PPro_50k.

Figure 3

IRI activity of polypeptides. (A) Dose-dependent IRI activity of poly(l-proline) and poly(sarcosine). (B) Example cryomicrographs from the “splat” assay after 30 min annealing at −8 °C in PBS for 10 mg mL^–1 of PPro_20k (left) and PSar_20k (right). (C) Example cryomicrographs in a sucrose sandwich (45 wt % sucrose) assay at −20 °C containing 10 mg mL^–1 of PPro_50k.

Figure 4

(A) Modified “sucrose sandwich” ice shaping assay images showing rectangular and blunt needle shape for 2 mg mL^–1 PPro_50k in 45 wt % sucrose solution before and after 1 h growth. (B) single crystal ice shaping using 2 mg mL^–1 PPro_50k showing formation of flower-shaped ice crystal as the ice crystal grows.