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Construction of multivalent homo- and hetero-functional ABO blood group glycoconjugates using a trifunctional linker strategy

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  • The design and synthesis of multivalent ligands displaying complex oligosaccharides is necessary for the development of therapeutics, diagnostics, and research tools. Here, we report an efficient conjugation strategy to prepare complex glycoconjugates with four copies of one or two separate glycan epitopes, providing 4–8 carbohydrate residues on a tetravalent poly(ethylene glycol) scaffold. This strategy provides complex glycoconjugates that approach the size of glycoproteins (15–18 kDa) while remaining well-defined. The synthetic strategy makes use of three orthogonal functional groups, including a reactive NHS-ester moiety on the linker to install the first carbohydrate epitope via reaction with an amine. A masked amine functionality on the linker is revealed after removal of a fluorenylmethyloxycarbonyl (Fmoc)-protecting group, allowing attachment to the NHS-activated PEG scaffold. An azide group in the linker was then used to incorporate the second carbohydrate epitope via catalyzed alkyne–azide cycloaddition. Using a known tetravalent PEG-scaffold (PDI, 1.025), we prepared homofunctional glycoconjugates that display four copies of lactose, the A-type II or the B-type II human blood group antigens. Using our trifunctional linker, we expanded this strategy to produce heterofunctional conjugates with four copies of two separate glycan epitopes. These heterofunctional conjugates included Neu5Ac, or '-sialyllactose, or 6'-sialyllactose as a second antigen. Using an alternative strategy, we generated heterofunctional conjugates with three copies of the glycan epitope and one fluorescent group (on average) using a sequential dual-amine coupling strategy. These conjugation strategies should be easily generalized for conjugation of other complex glycans. We demonstrate that the glycan epitopes of heterofunctional conjugates engage and cluster target BCR and CD22 receptors on B cells, supporting the application of these reagents for investigating cellular response to carbohydrate antigens of the ABO blood group system.

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    Article (Published)
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    Attribution-NonCommercial 4.0 International
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