Chemical and Enzymatic Synthesis of Sialylated Glycoforms of Human Erythropoietin

Abstract

Recombinant human erythropoietin (EPO) is the main therapeutic glycoprotein for the treatment of anemia in cancer and kidney patients.T he in-vivo activity of EPO is carbohydrate-dependent with the number of sialic acid residues regulating its circulatory half-life.E PO carries three Nglycans and thus obtaining pure glycoforms provides am ajor challenge.W eh ave developed ar obust and reproducible chemoenzymatic approach to glycoforms of EPO with and without sialic acids.E PO was assembled by sequential native chemical ligation of two peptide and three glycopeptide segments.The glycopeptides were obtained by pseudoprolineassisted 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 1:1c omplexes with recombinant EPO receptor.

Semisynthesis of homogeneous spike RBD glycoforms from SARS-CoV-2 for profiling the correlations between glycan composition and function

“…Preparation of sialylated glycopeptide (C336–K378) commenced with synthesis of the protected peptide 15 in which Boc-hydrazide was induced to prevent undesired conjugation during subsequent HATU-assisted aspartylation. Generation of glycopeptide 16 was achieved through pseudoproline-mediated one-flask aspartylation [ 33 , 37 , 38 ]. Coupling of the carboxylic acid at D343 with glycosyl amine 7 produced 16 with a 49% isolated yield.…”

Total synthesis of interleukin-2 via a tunable backbone modification strategy

“…Many proteins of biological interests have been chemically synthesized and used to correlate the protein structure to function. [14][15][16][17][18][19][20][21][22][23][24] In particular, protein chemical synthesis provides a unique means to generate proteins with tailor-made modications, which are difficult or impossible for expression systems. [25][26][27][28][29][30][31][32][33][34][35][36] Despite these achievements in protein chemical synthesis, the synthesis of proteins or peptides with aggregation-prone properties remains a challenging task and requires case by case analysis and study.…”

Chemical Synthesis Creates Single Glycoforms of the Ectodomain of Herpes Simplex Virus-1 Glycoprotein D

“…Current isolation techniques and gene expression technologies find it difficult to produce homogeneous gD. Chemical methods have been developed to prepare glycoproteins bearing homogeneous N -linked glycans, − for example, EPO, − Interleukin-17A, and SARS-CoV-2 Spike Receptor-Binding Domain . And the synthesis of large-sized proteins has now become achievable due to recent advancements in peptide synthesis methods and ligation techniques. − However, considering the sophisticated oligosaccharide structures, protein size and the absence of appropriate cysteine residues for ligation, the chemical synthesis of complex glycoproteins (over 200 amino acids in length) such as gD bearing complex N -linked glycan remains a formidable challenge .…”