Chemoenzymatic assembly of structurally defined heparan sulfate oligosaccharides reveals heparanase substrate plasticity and enables inhibitor design.

Heparanase (HPSE) is a promising therapeutic target, yet current heparin-derived inhibitors are limited by structural heterogeneity, poor selectivity, and off-target anticoagulant effects. Here, we synthesized a library of structurally defined heparan sulfate (HS) oligosaccharides via a robust chemoenzymatic strategy, enabling a systematic investigation of HPSE cleavage specificity. Our data confirm the minimal cleavable tetrasaccharide motif for HPSE and the preference for the endolytic site proximal to the nonreducing end in HS hexasaccharides. We demonstrate that a 2-O-sulfated iduronic acid (IdoA2S) residue confers susceptibility to HPSE cleavage on 6-O-nonsulfated HS oligosaccharides through compensatory interactions with the enzyme's heparin-binding domain (HBDs), and acts synergistically with 6-O-sulfated GlcNS residues to promote catalytic efficiency. HPSE exhibits an atypical exo-type cleavage at the nonreducing-terminal GlcA residue, dependent on sulfation patterns and chain length, challenging the traditional classification of HPSE as a strictly endo-acting β-glucuronidase. Leveraging insights into substrate plasticity, we rationally designed highly sulfated HS oligosaccharides, each featuring a disfavored terminal GlcA motif linked to an IdoA2S-enriched sequence. A 3-O-sulfated HS nonasaccharide 28 exhibited sub-micromolar HPSE inhibitory potency (IC50 = 0.761 μM) and is expected to minimize anticoagulant liabilities by lacking the canonical antithrombin-binding sequence. Collectively, this work elucidates the molecular determinants of HPSE substrate plasticity and provides a structure-based foundation for the rational design of next-generation anti-metastatic agents with improved selectivity and safety profiles.