A synthetic, sulfation-defined heparan sulfate (HS) / heparin-like tetrasaccharide bearing the GlcAβ1-4GlcNAcα1-4GlcAβ1-4GlcNAc sequence with 6-O-sulfation on the non-reducing-end GlcNAc only. Used as a probe to interrogate the contribution of single-site sulfation to HS/heparin protein interactions. Heparan sulfate (HS) and heparin are sulfated glycosaminoglycans found as components of proteoglycans. HS, in close proximity to cell surfaces and extracellular matrix proteins, plays essential physiological roles binding hundreds of proteins, and may play a role in SARS-CoV-2 infection through virus binding to ACE2; heparin is mainly found in mast cell secretory granules. Synthetic, structurally defined HS/heparin fragments are valuable probes for sulfation-pattern-dependent protein binding, anticoagulant and growth-factor signaling research, and as standards for analytical and structure-activity studies.