A systematic, efficient means of producing diverse libraries of
asymmetrically branched N-glycans is needed to investigate the
specificities and biology of glycan binding proteins. To that end, we describe a
core pentasaccharide that at potential branching positions is modified by
orthogonal protecting groups to allow selective attachment of unique saccharide
moieties by chemical glycosylation. The appendages were selected in such a way
that the antenna of the resulting deprotected compounds could be selectively
extended by glycosyltransferases to give libraries of asymmetrical
multi-antennary glycans. The power of the methodology was demonstrated by the
preparation of a series of complex oligosaccharides that were printed as
microarrays and screened for binding to lectins and influenza-virus
hemagglutinins, which showed that recognition is modulated by presentation of
minimal epitopes in the context of complex N-glycans.
Glycoamino
acid analogues of the Thomsen–Friedenreich antigen
disaccharide, where the 4′ and 4″ hydroxyl groups were
substituted with fluorine or hydrogen, were synthesized and incorporated
into the asialylated antiproliferative factor (as-APF), a biologically active form of APF, a glycopeptide found in
the urine of patients with interstitial cystitis. Various strategies
were employed to incorporate the fluorine atom at the 4-positions
of either the galactose or N-acetylgalactosamine
unit of the disaccharide antigen, based on stereochemistry and reactivity.
These glycopeptides were evaluated in antiproliferative assays on
both primary normal bladder epithelial cells and T24 bladder carcinoma
cells. Unlike many previously published substitutions to APF, mono-4′-fluorination
of the GalNAc residue did not affect the activity, whereas fluoro-derivatives
of the galactose 4″-position or both 4′ and 4″
hydroxyls showed a reduced potency relative to the monosubstituted
GalNAc derivative. A fourth compound where the 4″ position
of galactose was deoxygenated showed a lower potency than the parent
and monosubstituted compounds. These results suggest that specific
substitutions in the sugar moieties in the APF can be tolerated, and
the glycomimetic design of APF analogues can include fluorine in the
GalNAc sugar of the disaccharide.
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