Using Nlinked Glycosylation to Stabilize Bivalirudin Marcus Van

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 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), iso. Asp (β-Asp), and Glc. NAc-Asn replaced Asn, respectively. A glycoamino acid, Fmoc-Asn(Glc. NAc)-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 2 drug and render the drug ineffective. (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. Results The needed Glc. NAc-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). Discussion and Conclusion References (1) Rev Cardiovasc Med 2007, 8 Suppl 3, S 9 -17. (2) Journal of Pharmaceutical Sciences 2017, 106 (5), 1322 -1330. (3) Bioorg. Med. Chem. Lett. , 2011, 21, 4973– 4975. (4) Org. Biomol. Chem. , 2014, 12, 913– 918. • The Asp and iso. Aspcontaining 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 Acknowledgement We are grateful to the Research and Scholarship Office and Chemistry Department at Dordt College for the funding support.