Facile Preparation of a Glycopolymer Library by PET-RAFT Polymerization for Screening the Polymer Structures of GM1 Mimics

Commercialized oligosaccharides such as GM1 are useful for biological applications but generally expensive. Thus, facile access to an effective alternative is desired. Glycopolymers displaying both carbohydrate and hydrophobic units are promising materials as alternatives to oligosaccharides. Prediction of the appropriate polymer structure as an oligosaccharide mimic is difficult, and screening of the many candidates (glycopolymer library) is required. However, repeating polymerization manipulation for each polymer sample to prepare the glycopolymer library is time-consuming. Herein, we report a facile preparation of the glycopolymer library of GM1 mimics by photoinduced electron/energy transfer-reversible addition–fragmentation chain-transfer (PET-RAFT) polymerization. Glycopolymers displaying galactose units were synthesized in various ratios of hydrophobic acrylamide derivatives. The synthesized glycopolymers were immobilized on a gold surface, and the interactions with cholera toxin B subunits (CTB) were analyzed using surface plasmon resonance imaging (SPRI). The screening by SPRI revealed the correlation between the log P values of the hydrophobic monomers and the interactions of the glycopolymers with CTB, and the appropriate polymer structure as a GM1 mimic was determined. The combination of the one-time preparation and the fast screening of the glycopolymer library provides a new strategy to access the synthetic materials for critical biomolecular recognition.


Setup of the equipment for PET-RAFT polymerization at open-air condition.
The equipment for PET-RAFT polymerization was assembled using a regulated power supply, a circuit board, and LEDs. Each LED bulb was fitted into a 96-well plate with a hole diameter of 4.5 mm for each well to serve as a light source. Two circuits, where four LEDs were connected in series, were connected in parallel to the power supply. The voltage and current of the regulated power supply were set as 14 V and 0.05 A, respectively.

Synthesis of galactose acrylamide (GalAAm)
TBTA (265 mg, 0.5 mmol), galactose azide (1.02 g, 5.0 mmol), BtnAAm (615 mg, 5.0 mmol), and CuSO4 (80 mg, 0.5 mmol) were dissolved in MeOH (25 mL) / H2O (25 mL) mixture. The oxygen was removed by bubbling nitrogen. L-Asc-Na (200 mg, 1.0 mmol) was added and stirred at 30 °C for 24 h under nitrogen atmosphere. The solution was concentrated under reduced pressure, and the precipitate was filtered. The crude product was purified by reverse-phase chromatography (Biotage SNAP ULTRA C18, gradient from water to methanol). The fraction containing the product was concentrated under reduced pressure and stirred with a metal scavenger (2.5 g) at room temperature for 24 h. After removal of metal scavenger of SiliaMets by filtration, the solution was obtained by freeze-drying (893 mg, 55%).

Synthesis of N-butylacrylamide (ButylAAm)
Butyl amine (300 mg, 3.0 mmol) and N,N-diisopropylethylamine (0.63 mL, 3.6 mmol) were dissolved in dry dichloromethane (6 mL) and stirred in ice bath. Acryloyl chloride (0.29 mL, 3.6 mmol) was slowly dropped into the solution and the mixture was stirred for 10 h at room temperature. The progress of the reaction was confirmed by TLC (EtOAc : hexane = 2 : 1, UV). The reactant was washed by saturated brine once. The organic phase was dried by MgSO4, filtered and concentrated under reduced pressure. The crude product was purified by silica column chromatography (EtOAc: hexane = 2 : 1) to give N-butyl acrylamide as white solid (203 mg, 53%). 1

Synthesis of N-cyclohexyl acrylamide (CyHexAAm)
Cyclohexyl amine (292 mg, 2.9 mmol) and N,N-diisopropylethylamine (0.61 mL, 3.5 mmol) were dissolved in dry dichloromethane (5.2 mL) and stirred in ice bath. Acryloyl chloride (0.26 mL, 3.2 mmol) was slowly dropped into the solution and the mixture was stirred for 10 h at room temperature.
The progress of the reaction was confirmed by TLC (EtOAc : hexane = 2 : 1, UV). The reactant was washed by saturated NaHCO3(aq) once. The organic phase was dried by MgSO4, filtered and concentrated under reduced pressure. The crude product was purified by silica column chromatography (EtOAc: hexane = 2: 1) to give N-cyclohexyl acrylamide (357 mg, 80%). 1    137 mm NaCl, 2.68 mm KCl) was flew through (0.1 mL/min), and SPRI reflectivity change (defined as "SPRI signal") was monitored until the SPRI signal was stable. Then, protein solution with a certain concentration was injected with flow rate of 0.1 mL/min in all experiments, and the SPRI signal was monitored. In the measurement, the SPRI signal was regarded as the amount of protein adsorption.
The binding constants of CTB were calculated with the Langmuir isotherm using the SPRI signals ∆ = ∆ max

1+
(1) S7 ΔR, ΔRmax, c, and Ka are the SPRI signal, the maximum SPRI signal, the protein concentration, and the binding constant, respectively. Based on eq 1, the plots of the SPRI signals were analyzed by nonlinear regression to derive the binding constants.