Probing the Mechanism of Action of Small-Molecule Ice Recrystallization Inhibitors Using Proton Nuclear Magnetic Resonance Relaxation

N -2-Fluorophenyl- d -gluconamide (2FA) improves the recovery and function of cryopreserved biological materials by inhibiting ice recrystallization. However, as for many small-molecule ice recrystallization inhibitors, the mechanism of action of 2FA is not well-understood. In this study, the IC 50 of 2FA for ice recrystallization was determined to be 3.5 mM (95% CI [3.41–3.52]). 1 H transverse and longitudinal relaxations were then characterized by NMR at 2FA concentrations from 0 to 10 mM and at temperatures between −15 °C and +30 °C. Corresponding activation energy of water molecule motion ( E A H2O ) was calculated, showing that at each concentration 2FA did not affect E A H2O in the solid state, whereas in the liquid state E A H2O was significantly higher with 2FA than for pure water. Therefore, 2FA is excluded from the ice lattice upon freezing and concentrated in the interstitial liquid phase. This restricts the migration of water molecules between ice crystals via the liquid phase, inhibiting ice recrystallization.