Amino acid-functionalised trehalose for multifunctional cryopreservation

Despite the critical importance of cryopreservation to medical science, progress is hindered by the toxicity of conventional cryoprotective agents (CPAs), in particular dimethyl sulfoxide (DMSO). Inspired by the natural accumulation of protective amino acids and trehalose during cold stress, we report the synthesis and evaluation of 6,6'-trehalose diesters of glycine (1a), L-alanine (1b), and L-proline (1c) as novel CPAs that address multiple modes of cryoinjury. Differential scanning calorimetry (DSC) confirmed favourable sub-zero glass transition temperatures (T_g) for these compounds, with values of up to -25 °C for 1c, consistent with enhanced vitrification capacity. Diesters exhibited comparable ice recrystallisation inhibition (IRI) activity to trehalose, despite sterically demanding modification at the 6,6'-positions. Modified freeze-float assays revealed controlled ice nucleation at elevated sub-zero temperatures, potentially enabling protective cellular dehydration before intracellular ice formation. Diesters 1a-c exhibited direct radical-scavenging activity, addressing a major limitation of native trehalose in cryopreservation. Molecular dynamics (MD) simulations provided complementary molecular-level insight into hydration behaviour, indicating highly localised hydration shells that disrupt extended water networks. Evaluation with mammalian cells (HaCaT) showed that these compounds possess favourable cytocompatibility, with significantly lower cytotoxicity than DMSO and trehalose at multiple time points. Cryopreservation trials revealed diesters 1a-c conferred measurable cryoprotection, despite lower overall recovery relative to 10 wt% DMSO. These findings establish bio-inspired functionalisation of trehalose as a promising strategy toward DMSO-free cryopreservation.