1. Discussion and Conclusion
Using N-linked Glycosylation to Stabilize Bivalirudin
Marcus Van Engen, Bennett Harmelink, Lilly Smith, Jenna Veenstra, Victoria Cast, and Joshua Zhu
Department of Chemistry, Dordt College, Sioux Center, IA 51052
Abstract
Results
The needed GlcNAc-Asn-9 and modified glycine have been synthesized in five and two steps respectively in gram scale (Scheme 1 and 2) and characterized by NMR according to reported procedures.3-4 Our four target peptides have also been successfully synthesized and identified by ESI-MS and HPLC. Moreover, the modified glycine showed that it can prevent the side reaction (aspartimide reaction) to help us obtain our target peptides (compound 3 and 4, Figure 2).
N-linked glycosylation was applied to bivalirudin to increase its stability. 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 glycobivalirudin, and a protected glycine, Fmoc-Gly(Dmb)-OH, was synthesized and applied to synthesize the α and β-Asp bivalirudin, preventing an aspartimide side reaction.
Introduction
Bivalirudin is an FDA approved direct thrombin inhibitor (DTI) used to prevent blood clotting during invasive cardiovascular procedures.1 Bivalirudin is a relatively unstable peptide drug. Deamidation of bivalirudin’s Asp-9 residue causes two major impurities (α and β-Asp), which occur in storage, manufacturing, and administration of the drug and render the drug ineffective.2 (Figure 1) N-linked glycosylation, a post-translational modification process in natural proteins, predominantly modifies Asn residues, stabilizing them from deamidation reactions. Thus, we chemically installed N-linked glycosylation onto the Asn-9 residue of Bivalirudin. This project has three goals: 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 are equivalent to normal bivalirudin.
Discussion and Conclusion
The Asp and isoAsp-containing chains will be used as standards to identify the HPLC peaks of deamidated bivalirudin.
Bivalirudin and glycobivalrudin will be placed in water-for-injection solutions to compare stability.
Glycobivalirudin’s thrombin inhibition will be tested with a fluorometric assay
References
(1) Rev Cardiovasc Med 2007, 8 Suppl 3, S9-17.
(2) Journal of Pharmaceutical Sciences 2017, 106 (5),
(3) Bioorg. Med. Chem. Lett., 2011, 21, 4973–4975.
(4) Org. Biomol. Chem., 2014, 12, 913–918.
Acknowledgement
We are grateful to the Research and Scholarship Office and Chemistry Department at Dordt College for the funding support.