Bivalirudin is an FDA approved direct thrombin inhibitor (DTI) used to prevent blood clotting during invasive cardiovascular procedures such as percutaneous coronary intervention.1 Two major impurities of bivalirudin are Asp-9 (α and β-Asp), caused by deamidation of the Asn-9 residue, which occurs in manufacturing, storage, and administration of the drug.2 N-linked glycosylation, a post-translational modification process in natural proteins, predominantly modifies Asn residues, which can stabilize them from deamidation reactions. Herein, we are chemically installing N-linked glycosylation onto the Asn-9 residue of Bivalirudin to reduce or eliminate Asp-9 impurities. Therefore, this project had three tasks: first, to demonstrate that N-linked glycosylation of the Asn-9 residue increases bivalirudin’s stability; secondly, to identify α and β-Asp impurities and their ratio formed from the deamidation reaction; thirdly, to verify that the inhibitory effects of the glycosylated bivalirudin (glycobivalirudin) are equivalent to normal bivalirudin. To achieve these goals, four peptide chains have been successfully synthesized via solid phase peptide synthesis: bivalirudin and three peptides that differ at only the ninth residue, where Asp (α-Asp), isoAsp (β-Asp), and GlcNAc-Asn replaced Asn, respectively. A glycoamino acid, Fmoc-Asn(GlcNAc)-OH, was synthesized as a building-block of the glycobivalirudin peptide, and a protected glycine, Fmoc-Gly(Dmb)-OH, was synthesized and applied in the syntheses of α and β-Asp bivalirudin, preventing an aspartimide side reaction, which makes the syntheses of these peptides difficult. The Asp and isoAsp containing chains, equal in structure to the deamidation products of bivalirudin, will be used as standards to identify the HPLC peaks of degraded bivalirudin. Bivalirudin and the GlcNAc-Asn-containing chain, glycobivalrudin, will be placed in water-for-injection solutions to examine stability. Glycobivalirudin will be further tested with a thrombin inhibition kit. Future projects may explore the efficacy and stability of glycobivalirudin in cytoplasm and animals, with a long-term goal of using this glycosylated drug on patients.
1. Lepor, N. E., Anticoagulation for acute coronary syndromes: from heparin to direct thrombin inhibitors. Rev Cardiovasc Med 2007, 8 Suppl 3, S9-17.
2. Zupančič, O.; Grießinger, J. A.; Lam, H. T.; Bernkop-Schnürch, A., Storage Stability of Bivalirudin: Hydrophilic Versus Lipophilic Solutions. Journal of Pharmaceutical Sciences 2017, 106 (5), 1322-1330.