Heparan sulfate tetrasaccharide 2 was efficiently prepared in seven steps through chemoenzymatic synthesis. A monosaccharide 5, N-acetyl-alpha-D-glucosamine- O-methylglycoside (GlcNAc-OMe), was successfully used as an acceptor in a heparosan synthase (pmHS2)-catalyzed glycosylation reaction. This avoided the multi-step synthesis of a more complex disaccharide acceptor 3, greatly simplifying the route to tetrasaccharide target 2. This approach provides a critical tetrasaccharide intermediate for subsequent chemoenzymatic transformation to fondaparinux and its analogues. (C) 2019 Elsevier Ltd. All rights reserved.