Chemical and Enzymatic Synthesis of Sialylated Glycoforms of Human Erythropoietin

• Published in: Angewandte Chemie International Edition Vol. 60; no. 49; pp. 25922 - 25932
• Main Authors: Hessefort, Hendrik, Gross, Angelina, Seeleithner, Simone, Hessefort, Markus, Kirsch, Tanja, Perkams, Lukas, Bundgaard, Klaus Ole, Gottwald, Karen, Rau, David, Graf, Christopher Günther Franz, Rozanski, Elisabeth, Weidler, Sascha, Unverzagt, Carlo
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 01.12.2021; John Wiley and Sons Inc
• Edition: International ed. in English
• Subjects: Anemia; Antibiotics; beta-D-Galactoside alpha 2-6-Sialyltransferase; Carbohydrates; Chemical synthesis; Enzymatic synthesis; Erythropoietin; Erythropoietin - chemistry; Erythropoietin - metabolism; Glycopeptides; Glycoproteins; Glycosylation; Humans; Molecular Structure; N-Acetylneuraminic Acid - biosynthesis; N-Acetylneuraminic Acid - chemical synthesis; N-Acetylneuraminic Acid - chemistry; native chemical ligation; oligosaccharide; Photobacterium - enzymology; Polysaccharides; receptor; Segments; Sialic acids; Sialyltransferases - metabolism; oligosaccharide; glycopeptides; glycoproteins; receptor; native chemical ligation
• ISSN: 1433-7851; 1521-3773; 1521-3773
• DOI: 10.1002/anie.202110013

Recombinant human erythropoietin (EPO) is the main therapeutic glycoprotein for the treatment of anemia in cancer and kidney patients. The in‐vivo activity of EPO is carbohydrate‐dependent with the number of sialic acid residues regulating its circulatory half‐life. EPO carries three N‐glycans and thus obtaining pure glycoforms provides a major challenge. We have developed a robust and reproducible chemoenzymatic approach to glycoforms of EPO with and without sialic acids. EPO was assembled by sequential native chemical ligation of two peptide and three glycopeptide segments. The glycopeptides were obtained by pseudoproline‐assisted Lansbury aspartylation. Enzymatic introduction of the sialic acids was readily accomplished at the level of the glycopeptide segments but even more efficiently on the refolded glycoprotein. Biological recognition of the synthetic EPOs was shown by formation of 1:1 complexes with recombinant EPO receptor. Two glycoforms of human erythropoietin (EPO) were synthesized by sequential native chemical ligation combining chemical and enzymatic methods. The sialylated glycoform EPO S was assembled stepwise from sialoglycopeptides but more rapidly by direct sialylation of the asialo glycoform EPO A. The native fold was evident from CD spectra, HR‐MS, and binding to the human EPO receptor (EPOR).