Tosylation-mediated uronic acid knockout from cryoprotective FucoPol revealed the importance of polyanionicity in ice growth disruption

Negative formal charge is a critical structural trait for the expression of cryoprotection due to its strong ice growth disruption at the growing ice facet. Polyanionic polysaccharides containing uronic acids (UA) in their structure are therefore often associated with psychrophilic-like traits, such as defense against cryoinjury. However, their practical biological post-thaw benefit is convoluted amongst other contributing factors, including hydrophilic-hydrophobic balance, molecular weight, rheological properties, and structural conformation. Here, we leveraged ubiquitous (31.7% m.d.w.) and COOH-targeted (63.4% m.d.w.) leaving-group SN2 tosylation procedures, coupled with clean purification methodologies (high-MW membrane dialysis), to synthesize UA-free variants (FP-OTs18, FP-OTs96) of the cryoprotective, UA-containing polysaccharide FucoPol (FP). Tosylation was confirmed by ATR-FTIR (1602.3 cm-1 shift to 1627.9 cm-1) and 1H-NMR (brs,{delta} 7.92 ppm; brs, 7.53 ppm). Tosylated derivatives exhibited reduced solution conductivity ({Omega}=300 {micro}S/cm vs. ~800 {micro}S/cm), unchanged molecular weight (Mw), and retained biocompatibility. However, the substitution of hydrophilic, low-steric hindrance carboxyl groups with hydrophobic, high steric hindrance tosyl groups promoted a 30-40% reduction in zero-shear viscosity (0) and precluded the formation of a hypothermic gel-state due to loss of inter-chain non-covalent interactions, leading to a drastic loss of cryoprotective activity (10-70% of the original post-thaw viability observed for native FucoPol). The cryobiologically antagonistic effect of FP-OTs18 and FP-OTs96 reveals that UAs are a key player in polysaccharide-based cryopreservation, not only due to standalone beneficial polyanionicity, but also because their presence largely contributes to the expression of agonistic structural traits that elicit a cryoprotective response.