Site-directed conjugation of "clicked" glycopolymers to form glycoprotein mimics: binding to mammalian lectin and induction of immunological function

Abstract

Synthesis of well-defined neoglycopolymer-protein biohybrid materials and a preliminary study focused on their ability of binding mammalian lectins and inducing immunological function is reported. Crucial intermediates for their preparation are well-defined maleimide-terminated neoglycopolymers (M(n) = 8-30 kDa; M(w)/M(n) = 1.20-1.28) presenting multiple copies of mannose epitope units, obtained by combination of transition-metal-mediated living radical polymerization (TMM LRP) and Huisgen [2+3] cycloaddition. Bovine serum albumin (BSA) was employed as single thiol-containing model protein, and the resulting bioconjugates were purified following two independent protocols and characterized by circular dichroism (CD) spectroscopy, SDS PAGE, and SEC HPLC. The versatility of the synthetic strategy presented in this work was demonstrated by preparing a small library of conjugating glycopolymers that only differ from each other for their relative epitope density were prepared by coclicking of appropriate mixtures of mannopyranoside and galactopyranoside azides to the same polyalkyne scaffold intermediate. Surface plasmon resonance binding studies carried out using recombinant rat mannose-binding lectin (MBL) showed clear and dose-dependent MBL binding to glycopolymer-conjugated BSA. In addition, enzyme-linked immunosorbent assay (ELISA) revealed that the neoglycopolymer-protein materials described in this work possess significantly enhanced capacity to activate complement via the lectin pathway when compared with native unmodified BSA.

Figure 1

SDS-PAGE [Coomassie staining (left) and UV excitation at λ = 302 nm (right)] for the conjugation reaction of BSA with 4a: (a) MW markers; (b) native BSA; (c) glycopolymer 4a; (d) BSA with 10 equiv of 4a: conjugation mixture; (e) 10 purified by SEC followed by affinity chromatography.

Figure 2

BSA + maleimide glycopolymer 4a (10 equiv). (Left) Purification of conjugate 10 through an immobilized Concanavalin A column before eluting with 1.0 M mannose solution. (Right) SEC analysis of the conjugation reaction mixture (purple) and the final purified product 10. UV detection (λ = 280 nm) was employed.

Figure 3

BSA (10 equiv) + maleimide glycopolymer 4a. FPLC anion-exchange chromatography purification of the conjugate 10 using Source Q column in 20 mM TRIS buffer at pH 9.0 as the mobile phase. A gradient of NaCl (pink dotted line) was used in order to elute the protein-based products.

Figure 4

Surface plasmon resonance analysis of immobilized BSA–glycopolymer conjugate 10 with fluid phase mannose binding lectin (MBL) (a), and immobilized MBL with fluid phase BSA–glycopolymer conjugate (b). Due to the naturally heterogeneous nature of MBL oligomers, concentrations of fluid-phase MBL on immobilized BSA–conjugate are indicated assuming a molecular weight of 75 kD. (b) An average molecular weight of 100 kDa is assumed for indications of fluid-phase BSA–conjugate.

Figure 5

Detection of complement system activation. BSA–conjugate 10, BSA, and yeast mannan were immobilized on microwell plates and exposed to diluted human serum. Values indicate the level of activated complement C9 deposition resulting from positive cascade triggering, as determined by a subsequent ELISA-style assay involving p-nitrophenol detection at λ = 405 nm. ELISA units obtained as direct absorbance at 405 nm values with reagent blank values deducted

Scheme 1

Synthesis of the Glycoprotein Mimic Employed in This Study

Scheme 2

^a a Reagents and conditions: (a) CuSO₄, sodium ascorbate, methanol/H₂O (1:1 v/v), ambient temperature; (b) N-(ethyl)-2-pyridylmethanimide, Cu(I) Br, rhodamine B methacrylate 9, initiator 8, CH₃OH/H₂O 5:2 (v/v), ambient temperature; (c) toluene reflux, 18 h; (d) N-(ethyl)-2-pyridylmethanimide, Cu(I) Br, initiator 8, rhodamine B methacrylate 9, toluene, 30 °C; (e) TBAF·3H₂O, THF, 0 °C to ambient temperature, 12 h; (f) (i) 1, (PPh₃)₃CuBr, Et₃N, DMSO, ambient temperature, 2 days; (ii) toluene reflux, 18 h.

Scheme 3

Synthesis of the Glycoprotein Mimic 10 Obtained from Neoglycopolymer 4a

Scheme 4

^a Reagents and conditions: (a) α-2′-azidoethyl-d-mannopyranoside 1/β-2′-azidoethyl-d-galactopyranoside 28, (PPh₃)₃CuBr, Et₃N, DMSO, ambient temperature, 2 days; (b) reduced pressure, 80 °C, solid state, 12 h; (c) BSA (10 equiv), DMSO:PBS (50 mM, pH 7.0).

Scheme 5

^a Reagents and conditions: (a) toluene reflux, 18 h; (b) BSA (10 equiv) DMSO:PBS (50 mM, pH 7.0).