Structural Superimposition

Abstact

Bivalirudin is a 20-residue synthetic peptide that functions as a bivalent thrombin inhibitor by simultaneously engaging thrombin's active site and exosite I. Despite its widespread clinical use, comprehensive NMR-based characterization of bivalirudin and its process-related impurities has not been reported. Here, we present the first complete heteronuclear NMR analysis of bivalirudin and two major impurities-[Asp9]-bivalirudin (a deamidation-related impurity standard) and [desGlu13]-bivalirudin (a synthesis deletion)-at natural isotopic abundance. Using a combination of 1D and 2D (1)H, (13)C, and (15)N NMR experiments in DMSO-d(6) and aqueous solution, we achieved near-complete backbone and side-chain assignments, now deposited in the BMRB. We demonstrate that 2D heteronuclear amide maps can unambiguously differentiate the parent peptide from its process impurities. Additionally, we report the first solution-state NMR structure of free bivalirudin, revealing a compact architecture wherein the active site- and exosite-binding segments are spatially proximate, potentially reflecting a pre-binding intermediate. This study establishes a practical spectral reference framework and simplified 2D heteronuclear spectral fingerprints for structural integrity assessment and impurity tracking of bivalirudin.