Glycans Equipped for Conjugation & Immobilization

Glycan-protein interactions play crucial roles in a myriad of biological processes including cellular communication and recognition, cell signaling, development and differentiation, immune responses, and pathogen interactions, for example. These interactions, where glycans act as ligands and form the basis of specific recognition by glycan binding proteins (lectins) are at the heart of glycobiology.

Studying glycan-protein interactions is of great interest. Many techniques to study binding require the glycan to be immobilized. This provides a multivalent presentation of the glycan where strong avidity interactions are required. Additionally, glycans are increasingly being used in therapeutic and diagnostic applications where the glycan is conjugated directly to another (bio)molecule, such as a protein, lipid, oligonucleotide, or drug. Sussex Research Laboratories has developed a portfolio of glycans that are equipped with various linkers (aglycones) that enable direct conjugation or immobilization to a variety of platforms. These linkers include:

Biotinylated glycans – The strong affinity of streptavidin/neutravidin for biotin has been exploited for decades. Our biotinylated glycoconjugate probes can be immobilized on chips, in wells, or on various other platforms that are coated with streptavidin/neutravidin.

Amine-functionalized linkers – Glycans equipped with amine functionalized linkers can be covalently coupled with N-hydroxy succinimide (NHS) activated esters to form amides.

Carboxylic acid functionalized linkers – The carboxylic acids can be activated to NHS, sulfo-NHS, or pentafluorophenyl esters and reacted with amine functionalized platforms or lysine reduces on proteins or peptides, for example.

Click chemistry substrates – (i) Azides - Glycans equipped with azide functionalized linkers can be covalently coupled to terminal alkynes via copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) or to dibenzocyclooctyne (DBCO) using copper-free click chemistry. (ii) DBCO – Glycans equipped with DBCO can be covalently coupled to azide functionalized platforms using copper-free click chemistry. (iii) Terminal alkynes – Glycans equipped with terminal alkynes can be coupled to azide functionalized platforms using CuAAC.

Maleimide functionalized linkers – The maleimide group can be readily reacted with thiol groups. For instance, the maleimide can be used to conjugate glycans onto proteins by reaction with reactive thiols on cysteines.

Oxazolines – Structures with a terminal GlcNAc oxazoline moiety are substrates for endo-β-N-acetylglucosaminidases
and can be used to remodel the N-linked glycans on monoclonal antibodies (mAb).

Thiol functionalized linkers – The thiol group can be reacted with other thiol modified substrates to form disulfides or can be reacted with maleimides to form conjugates.

These glycan reagents may be used to:

• Study glycan-binding protein interactions via glycan arrays, Surface Plasmon Resonance (SPR), or Bio-Layer Interferometry (BLI).
• Visualize lectin-expressing cells in vitro using fluorescent, streptavidin beads coated with specific glycans.
• Visualize lectin-expressing cells in tissues.
• Purify lectins via affinity chromatography.
• Targeted therapeutic or diagnostic delivery.

The glycan ligands for conjugation and immobilization can also incorporate a variety of defined spacers between the glycan and functional group. These can include a short alkyl spacer such as a propyl (C3) unit or a monodisperse (discrete), polyethylene glycol (PEG) chain such as tri-ethylene glycol (PEG3). PEG is a stable, amphiphilic molecule that can offset the hydrophobic nature of certain substituents such as biotin or DBCO.