Almost all cell surface and secreted proteins are modified by covalently-linked carbohydrate moieties, and
these so called glycans have been implicated as essential mediators of processes such as protein folding, cell
signaling, fertilization, embryogenesis, neuronal development, and the proliferation of cells and their
organization into specific tissues. Also, overwhelming data supports the relevance of glycosylation in pathogen
recognition, inflammation, innate immune responses, and the development of autoimmune diseases and
cancer. Progress in glycoscience is hampered by a lack of well-defined complex oligosaccharide standards
which are needed for the fabrication of the next generation of microarrays, for the development of analytical
protocols to determine exact structures of isolated glycans, for the elucidation of pathways of glycoconjugate
biosynthesis, and as immunogens to produce MABs for glycoprotein isolation and visualization.
In this application, we propose to develop novel synthetic strategies that can readily provide large libraries of
symmetrical and asymmetrical N-glycans. The new methodologies will make use of readily available starting
materials and will be sufficiently standardized that many laboratories, including synthesis service units, can
adopt these methods. Furthermore, the synthetic principles of the new approaches can easily be applied to the
preparation of other classes of glycans such as O-linked glycans and human milk oligosaccharides (HMOs).
The new method will employ a symmetrical biantennary glycan that can easily be isolated from egg yolk.
Innovative enzymatic transformations will be developed to desymmetrize this glycan. Furthermore,
recombinant N-acetylglucosaminyltransferases (MGAT's) will be used to convert a bi-antennary glycan into tri-
and tetra-antennary structures. In the latter transformations, chemically modified UDP-GlcNAc donors will be
used to temporarily prevent an arm from enzymatic modification. The use of recently developed technology to
express recombinant mammalian glycosyltransferases will be a key feature of the new methodology. To
validate the robustness of the methodology, it will be applied to the preparation of a library of glycans derived
from human upper airway epithelial cells. The resulting glycans will be valuable for the development of the next
generation of glycan microarray to probe carbohydrate–protein interactions in the context of this cell type. The
scope of the chemoenzymatic methodology will be further extended by the development of methods that can
easily provide highly complex asymmetrical glycans that are modified by sulfate esters. An automation platform
will be developed to further increase the speed of chemoenzymatic synthesis using novel capture and release
strategies. Attention will focus on ion exchange and nickel-mediated histidine binding events for capture of
tagged oligosaccharides. A multi-channel liquid handling robot from Chemspeed equipped with a volumetric
dispensing system and lyophilizer will be employed as an automation tool. The latter methodology will, at first,
be employed for the preparation of O-linked glycans and human milk oligosaccharides.