Synthesis of high-mannose 1-thio glycans and their conjugation to protein

The oligosaccharides Man4 and Man5, substructures of the high-mannose glycans of HIV glycoprotein gp120, were synthesized with a terminal 1-thiomannopyranose residue. The anomeric thiol can be readily converted to an azidomethyl aglycone through reaction with dichloromethane and displacement with sodium azide. The resulting oligomannans were then conjugated to ubiquitin utilizing thiol alkylation or azide/alkyne reactive tethers of minimal length. By combining high efficiency conjugation reactions and a short tether, we sought to establish conjugation conditions that would permit high density clustering of oligomannans in conjugate vaccines that could produce antibodies able to bind gp120 and potentially neutralize virus. LC-UV-MS was used to separate, identify and quantify the ubiquitin glycoconjugates with differing degrees of oligomannan incorporation. Binding of the HIV protective monoclonal antibody 2G12 and concanavalin A to microtitre plates coated with glycoconjugates was measured by ELISA.