Exploiting Substrate Specificities of 6-O-Sulfotransferases to Enzymatically Synthesize Keratan Sulfate Oligosaccharides

[Image: see text] Keratan sulfate (KS) is a glycosaminoglycan that is widely expressed in the extracellular matrix of various tissue types, where it is involved in many biological processes. Herein, we describe a chemo-enzymatic approach to preparing well-defined KS oligosaccharides by exploiting the known and newly discovered substrate specificities of relevant sulfotransferases. The premise of the approach is that recombinant GlcNAc-6-O-sulfotransferases (CHST2) only sulfate terminal GlcNAc moieties to give GlcNAc6S that can be galactosylated by B4GalT4. Furthermore, CHST1 can modify the internal galactosides of a poly-LacNAc chain; however, it was found that a GlcNAc6S residue greatly increases the reactivity of CHST1 of a neighboring and internal galactoside. The presence of a 2,3-linked sialoside further modulates the site of modification by CHST1, and a galactoside flanked by 2,3-Neu5Ac and GlcNAc6S is preferentially sulfated over the other Gal residues. The substrate specificities of CHST1 and 2 were exploited to prepare a panel of KS oligosaccharides, including selectively sulfated N-glycans. The compounds and several other reference derivatives were used to construct a microarray that was probed for binding by several plant lectins, Siglec proteins, and hemagglutinins of influenza viruses. It was found that not only the sulfation pattern but also the presentation of epitopes as part of an O- or N-glycan determines binding properties.