Small Molecule Ice Recrystallization Inhibitors Enable Freezing of Human Red Blood Cells with Reduced Glycerol Concentrations

In North America, red blood cells (RBCs) are cryopreserved in a clinical setting using high glycerol concentrations (40% w/v) with slow cooling rates (~1°C/min) prior to storage at −80°C, while European protocols use reduced glycerol concentrations with rapid freezing rates. After thawing and prior to transfusion, glycerol must be removed to avoid intravascular hemolysis. This is a time consuming process requiring specialized equipment. Small molecule ice recrystallization inhibitors (IRIs) such as β-PMP-Glc and β-pBrPh-Glc have the ability to prevent ice recrystallization, a process that contributes to cellular injury and decreased cell viability after cryopreservation. Herein, we report that addition of 110 mM β-PMP-Glc or 30 mM β-pBrPh-Glc to a 15% glycerol solution increases post-thaw RBC integrity by 30-50% using slow cooling rates and emphasize the potential of small molecule IRIs for the preservation of cells.


General Experimental:
All anhydrous reactions were performed in flame-dried glassware under a positive pressure of dry argon. Air or moisture-sensitive reagents and anhydrous solvents were transferred with oven-dried syringes or cannulae. All flash chromatography was performed with E. Merck silica gel 60 (230-400 mesh). All solution phase reactions were monitored using analytical thin layer chromatography (TLC) with 0.2 mm pre-coated silica gel aluminum plates 60 F254 (E. Merck). Components were visualized by illumination with a short-wavelength (254 nm) ultraviolet light and/or staining (ceric ammonium molybdate, potassium permanganate, or phosphomolybdate stain solution). All solvents used for anhydrous reactions were distilled. Compounds below are in order of appearance in the manuscript. Intermediates that were not numbered in the manuscript received numbers beginning with 12. NMR spectra for novel compounds and final compounds assessed for IRI activity are provided. Figure S3. Synthesis of β-PMP-Gal (5) and β-PMP-Glc (6)

p-Methoxyphenyl-β-D-galactopyranoside (5)
Compound 5a (5.3 g, 11.6 mmol) was dissolved in a solution of sodium methoxide in methanol (25 mL) and stirred for one hour at room temperature. The solution was then neutralized with Amberlite® IR-120 (H + ) ionexchange resin and filtered. The filtrate was concentrated and the product was lyophilized to yield 5 as a white powder (3.3 g, 98%). Characterization data is consistent with that previously reported 1

p-Methoxyphenyl-β-D-glucopyranoside (6)
Compound 6a (6.75 g, 14.9 mmol) was dissolved in a solution of sodium methoxide in methanol (25 mL) and stirred for one hour at room temperature. The solution was then neutralized with Amberlite® IR-120 (H + ) ionexchange resin, filtered and concentrated. The filtrate was concentrated and the product was lyophilized to yield 6 as a white powder (4.1 g, 95%). 1

Ethyl 2,3,4,6-Tetra-O-Acetyl-1-thio-α-D-Mannopyranoside (17)
To a solution of D-mannose (2.0 g, 11.1 mmol) in dry pyridine (45 mL) was added acetic anhydride (26 mL) and the reaction mixture was stirred at 0 °C for 1 hour. A catalytic amount of 4-dimethylaminopyridine was added and the mixture was stirred at room temperature overnight. Ethanol was added and the solvent was evaporated under in vacuo and the residue was diluted in ethyl acetate, washed with sodium bicarbonate, water and brine. The mixture was dried with MgSO 4 , filtered and concentrated. The crude product was then added to a solution of

Ethyl 1-thio-α-D-Mannopyranoside (18)
Compound 17 (2.7 g, 6.9 mmol) was dissolved in a solution of sodium methoxide in methanol (10 mL) and stirred for one hour at room temperature. The solution was neutralized with Amberlite® IR-120 (H + ) ion-exchange resin, filtered and concentrated to yield 18 as a solid (1.5 g, 95%). Characterization data is consistent with that previously

p-Methylphenyl-β-D-glucopyranoside (9)
Compound 9a (124 mg, 0.28 mmol) was dissolved in a solution of sodium methoxide in methanol (5 mL) and stirred for one hour at room temperature. The solution was then neutralized with Amberlite® IR-120 (H + ) ionexchange resin, filtered and concentrated. The product was purified by recrystallization in hot ethyl acetate to afford 9 as a white powder (68 mg, 89%). Characterization data is consistent with that previously reported in the literature 11

p-Fluorophenyl-β-D-glucopyranoside (12)
Compound 12a (198 mg, 0.45 mmol) was dissolved in a solution of sodium methoxide in methanol (5 mL) and stirred for one hour at room temperature. The solution was then neutralized with Amberlite® IR-120 (H + ) ionexchange resin, filtered and concentrated. The filtrate was concentrated and the product was lyophilized to yield 12 as a white powder (120 mg, 98%
Column chromatography (4:1 hexanes/EtOAc) afforded 16a as a white powder (1.43 g, 30%). Characterization data is consistent with that previously reported in the literature 15