Synthesis of an End-to-End Protein-Glycopolymer Conjugate via Bio-Orthogonal Chemistry

We report the synthesis of an end-to-end protein-glycopolymer conjugate, namely, site-specific modification of recombinant thrombomodulin at the C-terminus with a chain-end-functionalized glycopolymer. Thrombomodulin (TM) is an endothelial membrane glycoprotein that acts as a major cofactor in the protein C anticoagulant pathway. To closely mimic the glycoprotein structural feature of native TM, we proposed a site-specific glyco-engineering of recombinant TM with a glycopolymer. Briefly, recombinant TM containing the epidermal growth factor (EGF)-like domains 4, 5, and 6 (rTM456) and a C-terminal azidohomoalanine was modified with a dibenzylcyclooctyne (DBCO) chain-end-functionalized glycopolymer via copper-free click chemistry to afford the end-to-end TM-glycopolymer conjugate. The TM glycoconjugation was confirmed with SDS-PAGE, Western blot, and protein C activation assay, respectively. The reported site-specific end-to-end protein glycopolymer conjugation approach facilitates uniform glycoconjugate formation via biocompatible chemistry and in high efficiency providing a rational strategy for generating an rTM-based anticoagulant agent.