An Automation Platform for the Chemoenzymatic Synthesis of Complex Sulfated and Branched Glycans

Diverse collections of well-defined glycans are needed to investigate the molecular mechanisms by which these biomolecules mediate biological and disease processes. Several automation approaches have been introduced to accelerate the enzymatic synthesis of complex glycans. These methodologies have, however, provided only relatively simple oligosaccharides due to limitations of glycosyl transferase selectivity. Here, we describe an automation platform that makes it possible, for the first time, to prepare in an automated fashion sulfated polylactosamines and asymmetric multiantennary complex N-glycans via sequential enzymatic and chemical reaction cycles. It integrates glycosyltransferase catalyzed glycosylations, the use of the unnatural sugar nucleotide donor 5'-diphosphate-2-deoxy-2-trifluoro-N-acetamido-glucose (UDP-GlcNHTFA), and chemical manipulations including base-mediated trifluoroacetamido (TFA) removal, azido transfer and azido reduction, tert-butyloxycarbonyl (Boc) protection, acid mediated deprotection, and amine acylation. The latter transformations are important for stop-and-go chemoenzymatic synthetic strategies in which unnatural monosaccharides are introduced to temporarily disable specific sites from enzymatic modification. It is shown that, due to the modular architecture of glycans, a limited number of glycosyl transferases can provide access to large numbers of structurally diverse glycans. In this study, only 11 recombinant human glycosyl- and sulfo transferases were employed to prepare highly complex glycans. Removal of the Nap tag can be performed by hydrogenation to give oligosaccharides that are ready for microarray printing or bioconjugation.