8EF4

Solution structural bundle of bivalirudin - a bivalent hirudin based thrombin inhibitor

Summary for 8EF4

• Entry DOI: 10.2210/pdb8ef4/pdb
• NMR Information: BMRB: 51618
• Descriptor: Bivalirudin (1 entity in total)
• Functional Keywords: thrombin inhibitor, bivalent, hirulog, blood clotting
• Biological source: Hirudo medicinalis (medicinal leech)
• Total number of polymer chains: 1
• Total formula weight: 2181.30

Authors

Mishra, S.H.,Bhavaraju, S. (deposition date: 2022-09-08, release date: 2022-10-05, Last modification date: 2026-08-12)

Primary citation

Mishra, S.H.,Kaur, A.,Vu, D.,Bhavaraju, S.
Comprehensive solution NMR characterization of bivalirudin, a direct thrombin inhibitor peptide, and its process impurities by heteronuclear spectral fingerprinting at natural isotopic abundance.
J Pharm Biomed Anal, 281:117684-117684, 2026

PubMed Abstract: 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 H, C, and N NMR experiments in DMSO-d₆ 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.

PubMed: 42542115
DOI: 10.1016/j.jpba.2026.117684

Experimental method

SOLUTION NMR