Potent inhibition of ice recrystallization by low molecular weight carbohydrate-based surfactants and hydrogelators

Abstract

Ice recrystallization inhibition (IRI) activity is a very desirable property for an effective cryoprotectant. This property was first observed in biological antifreezes (BAs), which cannot be utilized in cryopreservation due to their ability to bind to ice. To date, potent IRI active compounds have been limited to BAs or synthetic C-linked AFGP analogues (1 and 2), all of which are large peptide-based molecules. This paper describes the first example of low molecular weight carbohydrate-based derivatives that exhibit potent IRI activity. Non-ionic surfactant n-octyl-b-D-galactopyranoside (4) exhibited potent IRI activity at a concentration of 22 mM, whereas hydrogelator N-octyl-Dgluconamide (5) exhibited potent IRI activity at a low concentration of 0.5 mM. Thermal hysteresis measurements and solid-state NMR experiments indicated that these derivatives are not exhibiting IRI activity by binding to ice. For non-ionic surfactant derivatives (3 and 4), we demonstrated that carbohydrate hydration is important for IRI activity and that the formation of micelles in solution is not a prerequisite for IRI activity. Furthermore, using solid-state NMR and rheology we demonstrated that the ability of hydrogelators 5 and 6 to form a hydrogel is not relevant to IRI activity. Structurefunction studies indicated that the amide bond in 5 is an essential structural feature required for potent IRI activity.

Small molecule ice recrystallization inhibitors enable freezing of human red blood cells with reduced glycerol concentrations

“…However, D-glucose derivative b-PMP-Glc (6) was a very effective inhibitor of ice recrystallization and was significantly more active than b-PMP-Gal (5) and b-PMPMan (7). In fact, b-PMP-Glc (6) exhibited activity similar to the previously reported non-ionic surfactant b-Octyl-Gal (1) at equimolar concentration 25 . b-PMP-Glc (6) also exhibited comparable IRI activity to a 220 mM solution of sucrose, which is a common ingredient of many cryopreservation solutions 28 .…”

“…This trend was also observed in small molecules where the non-ionic pyranose-based surfactants containing a highly hydrated D-galactose head group (b-Octyl-Gal, 1, Fig. 1) was significantly more active than the surfactants containing the less hydrated D-glucose head group (b-Octyl-Glc, 2) 25 . The fact that the D-glucopyranose derivative b-PMP-Glc (6) exhibited potent IRI activity and was significantly more IRI active than D-galactopyranose derivative b-PMP-Gal (5) (Fig.…”

“…Prior work by our laboratory has demonstrated that amphiphilic small molecule carbohydrate derivatives (Fig. 1a) as well as some amino acid derivatives are very effective inhibitors of ice recrystallization [25][26][27] . In all of these compounds, the hydrophobic component of the molecule was an alkyl chain varying from 6-14 carbons in length.…”

“…Thus, NOGlc (4), which is highly IRI active at 0.5 mM (Fig. 1c) 25 was used in cryo-solutions at 2 mM or 5 mM concentrations while b-PMP-Glc (6) was used at 110 mM or 220 mM. NOGlc could not be assessed in the freezing assays at concentrations greater than 5 mM due poor solubility in aqueous solutions containing glycerol 25,49 .…”

“…As a consequence, there has been tremendous interest in designing compounds that can inhibit ice recrystallization for cryopreservation [16][17][18][19][20][21] . Our laboratory has recently reported the rational design and synthesis of large Clinked glycoconjugates [22][23][24] as well as small molecules [25][26][27] that inhibit the process of ice recrystallization. We hypothesize that inhibiting this process will greatly reduce cellular injury during cryopreservation and increase the post-thaw recovery of cells with specialized cellular functions 24,28 .…”