Site-Specific Tyrosine Sulfation of Triabin via Semi-Synthesis Enhances Thrombin Inhibition through Synergistic Multiple-Mode Interactions.

Herein, we report the semisynthesis of site-specifically sulfated triabin─a 142-amino-acid lipocalin protein─at Tyr124. Functional assays reveal that sulfation enhances anticoagulant activity by ∼5-fold, demonstrating that even rigid scaffolds can benefit from this modification. Modeling studies uncover a synergistic mechanism wherein the sulfate group engages in a hydrogen-bond network, electrostatic bridging, and charge-complementary interactions with thrombin exosite I, while the hydrophobic core (notably Phe106 and Val126) remains the primary driving force.