Using Automated Glycan Assembly (AGA) for the Practical Synthesis of Heparan Sulfate Oligosaccharide Precursors

Herein we report synthesis of complex heparan sulfate oligosaccharide precursors by automated glycan assembly using disaccharide donor building blocks. Rapid access to a hexasaccharide was achieved through iterative solid phase glycosylations on a photolabile resin using Glyconeer™, an automated oligosaccharide synthesiser, followed by photochemical cleavage and glycan purification using simple flash column chromatography

Chemoenzymatic Synthesis of Heparin Oligosaccharides with both Anti-factor Xa and Anti-factor IIa Activities

Heparan sulfate (HS) and heparin are highly sulfated polysaccharides. Heparin is a commonly used anticoagulant drug that inhibits the activities of factors Xa and IIa (also known as thrombin) to prevent blood clot formation. Here, we report the synthesis of a series of size-defined oligosaccharides to probe the minimum size requirement for an oligosaccharide with anti-IIa activity. The synthesis was completed by a chemoenzymatic approach involving glycosyltransferases, HS sulfotransferases, and C(5)-epimerase. We demonstrate the ability to synthesize highly purified N-sulfo-oligosaccharides having up to 21 saccharide residues. The results from anti-Xa and anti-IIa activity measurements revealed that an oligosaccharide longer than 19 saccharide residues is necessary to display anti-IIa activity. The oligosaccharides also exhibit low binding toward platelet factor 4, raising the possibility of preparing a synthetic heparin with a reduced effect of heparin-induced thrombocytopenia. The results from this study demonstrate the ability to synthesize large HS oligosaccharides and provide a unique tool to probe the structure and function relationships of HS that require the use of large HS fragments

Dissecting the substrate recognition of 3-O-sulfotransferase for the biosynthesis of anticoagulant heparin

Heparin is a polysaccharide-based natural product that is used clinically as an anticoagulant drug. Heparan sulfate 3-O-sulfotransferase (3-OST) is an enzyme that transfers a sulfo group to the 3-OH position of a glucosamine unit. 3-OST is present in multiple isoforms, and the polysaccharides modified by these different isoforms perform distinct biological functions. 3-OST isoform 1 (3-OST-1) is the key enzyme for the biosynthesis of anticoagulant heparin. Here, we report the crystal structure of the ternary complex of 3-OST-1, 3′-phosphoadenosine 5′-phosphate, and a heptasaccharide substrate. Comparisons to previously determined structures of 3-OST-3 reveal unique binding modes used by the different isoforms of 3-OST for distinguishing the fine structures of saccharide substrates. Our data demonstrate that the saccharide substrates display distinct conformations when interacting with the different 3-OST isoforms. Site-directed mutagenesis data suggest that several key amino residues, including Lys259, Thr256, and Trp283 in 3-OST-3 and Arg268 in 3-OST-1, play important roles in substrate binding and specificity between isoforms. These results deepen our understanding of the biosynthetic mechanism of heparan sulfate and provide structural information for engineering enzymes for an enhanced biosynthetic approach to heparin production

Chemoenzymatic Design of Heparan Sulfate Oligosaccharides*

Heparan sulfate is a sulfated glycan that exhibits essential physiological functions. Interrogation of the specificity of heparan sulfate-mediated activities demands a library of structurally defined oligosaccharides. Chemical synthesis of large heparan sulfate oligosaccharides remains challenging. We report the synthesis of oligosaccharides with different sulfation patterns and sizes from a disaccharide building block using glycosyltransferases, heparan sulfate C5-epimerase, and sulfotransferases. This method offers a generic approach to prepare heparan sulfate oligosaccharides possessing predictable structures

Toward the chemoenzymatic synthesis of heparan sulfate oligosaccharides: oxidative cleavage of p-nitrophenyl group with ceric ammonium salts

We have developed an efficient chemoenzymatic synthesis of heparan sulfate oligosaccharides employing the para-nitrophenyl (p-NP) β-glucuronide as an acceptor compatible with enzymatic elongation and one that significantly simplifies oligosaccharide purification on C-18 resin. Employing ceric ammonium nitrate as oxidative reagent to remove the p-NP group unexpectedly also removed the glucuronic acid residue at the reducing-end, affording a smaller oligosaccharide. The application of ceric ammonium sulfate allowed the removal of the p-NP without concomitant loss of the adjacent glucuronic acid offering a route to longer heparin sulfate oligosaccharide products