RAFT-based tri-component fluorescent glycopolymers: synthesis, characterization and application in lectin-mediated bacterial binding study

Abstract

A group of fluorescent statistical glycopolymers, prepared via reversible addition-fragmentation chain-transfer (RAFT)-based polymerizations, were successfully employed in lectin-mediated bacterial binding studies. The resultant glycopolymers contained three different monomers: N-(2-hydroxyethyl) acrylamide (HEAA), N-(2-aminoethyl) methacrylamide (AEMA) and N-(2-glyconamidoethyl)-methacrylamides possessing different pendant sugars. Low dispersities (≤1.32) and predictable degrees of polymerization were observed among the products. After the polymerization, the glycopolymers were further modified by different succinimidyl ester fluorophores targeting the primary amine groups on AEMA. With their binding specificities being confirmed by testing with lectin coated agarose beads, the glycopolymers were employed in bacterial binding studies, where polymers containing α-galactose or β-galactose as the pendant sugar were specifically bound by two clinically important pathogens Pseudomonas aeruginosa and Staphylococcus aureus, respectively. This is the first report of using RAFT-based glycopolymers in bacterial binding studies, and the ready access to tri-component statistical glycopolymers also warrants further exploration of their utility in other glycobiological applications.

Scheme 1

Illustration of the synthesis of fluorescent glycopolymers PMA-ALAEMA-Fluorescein containing β-galactoside as the pendant sugar

Fig. 1

Kinetic study for the RAFT polymerization of PMA-GAEMA. a Gel permeation chromatography traces of the copolymers indicating steady evolution of the copolymers with time. b Mn dispersities of the glycopolymers over time. ([M]₀:[CTA]:[Initiator] = 380:2:1; [M]₀ = 0.7 M; solvent, H₂O/DMF = 4:1)

Fig. 2

Gel permeation chromatography traces of RAFT-based glycopolymers. ([M]₀:[CTA]:[Initiator] = 380:2:1; [M]₀ = 0.7 M; solvent, H₂O/DMF = 4:1)

Fig. 3

Assigned ¹H- (a) and ¹³C-NMR (b) spectra (D₂O) for PMA-ALAEMA

Fig. 4

Gel permeation chromatography traces of PMA-ALAEMA with different DPs from copolymerizations using different amounts of chain transfer agent ([M]₀ = 0.7 M; solvent, H₂O/DMF = 4:1)

Fig. 5

Galanthus Nivalis lectin (GNL) coated agarose beads bind α-D-mannoside containing glycopolymers, but not those possessing α-D-galactoside. PMA-MAEMA-Fluorescein (3 μg) showed only a weak non-specific binding with GNL (a), in contrast to the strong binding between PMA-MGAEMA-Fluorescein and the beads (b). Pre-incubation of the beads with 1.0 mg of non-fluorescent PMA-MGAEMA for 20 min before adding 3 μg of PMA-MGAEMA-Fluorescein dramatically reduced the binding of the latter (c), but the pre-incubation with 1.0 mg of PMA-MAEMA didn’t show any competitive inhibition effects (d). Scale bar = 100 μm

Fig. 6

Representative lectin-mediated binding of bacteria to synthetic fluorescent glycopolymers. Fluorescence microscopy of a mixture of the binding tests: P. aeruginosa with PMA-MAEMA-Texas Red (100 μg) and S. aureus with PMA-ALAEMA-Fluorescein (100 μg). Scale bar = 100 μm

Fig. 7

S. aureus ATCC 25923 showed binding specificity towards glycopolymers containing β-galactose as the pendant sugar. Different fluorescent glycopolymers (100 μg) were used in corresponding binding test, and 100 μL of the final bacteria suspensions in PBS were used to measure their fluorescence intensities on the microplate reader (λex/λem = 490/520 nm, slit width = 10 nm)