| Heparan sulfate (HS), a highly sulfated polysaccharide, plays fundamental roles in a wide range of physiological and pathophysiological processes. The specific structure of HS determines its biological function. Various HS biosynthetic enzymes dictate the final structure of HS products. As an attractive target for medical and pharmaceutical research, synthesis of structurally defined HS oligosaccharides is one of the major challenges in the field of glycobiology. Enzymatic synthesis of HS represents a valuable complementary approach to chemical synthesis. The access to the unsulfated, unepimerized HS backbones is an essential step towards enzymatic synthesis of HS. In this research, we developed a chemoenzymatic approach for de novo synthesis of HS backbones with two bacterial glycosyltransferase, KfiA and PmHS2. KfiA is an N-acetylglucosaminyltransferase in Escherichia coli strain K5, while PmHS2 is a bifunctional enzyme with both N-acetylglucosaminyltransferase and D-glucuronyltransferase in Pasteurella multocida Types A, D, and F. Both of the recombinant proteins were prepared in large quantities. Characterization of KfiA reveals that its enzymatic activity is independent of the size of the acceptor substrates but a particular structure in the natural donor UDP-N-acetylglucosamine is required to bind KfiA. UDP-N-trifluoroacetylglucosamine, a derivative of UDP-N-acetylglucosamine, was recognized by KfiA and employed to synthesize unnatural HS backbones containing N-trifluoroacetylglucosamine residues. The synthesized oligosaccharides range from trisaccharide to undecasaccharide with a yield of 100 mug to milligram scales. The controlled N-sulfation on HS backbones was achieved by selective removal of N-trifluoroacetyl groups and a subsequent N-sulfation with N-sulfotransferase. The synthesized octasaccharide backbone with defined N-sulfation was further modified with a collection of HS biosynthetic enzymes and yielded a bioactive octasaccharide binding to antithrombin. The results of this study open a new approach for the de novo synthesis of structure-defined HS oligosaccharides. |
