Endohexosaminidase catalysed glycosylation with oxazoline donors: The development of robust biocatalytic methods for synthesis of defined homogeneous glycoconjugates

Glycoprotein remodelling represents a practical and potentially widely applicable method for the production of homogenous glycoconjugates bearing defined N-glycan structures, including therapeutic glycoproteins and monoclonal antibodies. Key to the remodelling process is the attachment of a defined oligosaccharide structure en bloc to GlcNAc residues at N-linked glycosylation sites. Endohexosaminidases (endo-β-N-acetyl-glucosaminidases, ENGases) are a class of enzyme that are capable of achieving this synthetic transformation with complete regio- and stereochemical control. In particular, the use of oxazolines as activated donor substrates for these enzymes greatly improves synthetic efficiencies as compared to transglycosylation using Asn-linked oligosaccharides. This article summarises recent work from within our laboratory focussing on the synthesis of a wide variety of N-glycan oxazolines and their use as substrates for endohexosaminidase-catalysed glycosylation. In particular, the problem of enzyme-catalysed competitive product hydrolysis may be countered, either by the use of structurally modified oxazoline donors, or by the production of mutant endohexosaminidase enzymes. The power of this methodology is exemplified by the production of a defined glycoform of ribonuclease B in a highly efficient fashion.