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.

Light‐Driven Self‐Recruitment of Biomimetic Semiconducting Polymer Nanoparticles for Precise Tumor Vascular Disruption

“…The expression levels of some related hematopoietic cytokines in the peripheral blood of mice after different treatments were first evaluated through enzyme‐linked immunosorbent assay analysis. These hematopoietic cytokines included: erythropoietin (EPO), which regulates the growth and differentiation of erythrocytes; [ 26 ] thrombopoietin (TPO), which regulates megakaryocyte production and maintains the quiescence of hematopoietic stem cells during hematopoiesis; [ 27 ] and granulocyte colony‐stimulating factor (G‐CSF), which is secreted by a variety of cancers and is responsible for the production, differentiation, and expansion of myeloid cells in the spleen. [ 28 ] The concentrations of EPO and TPO in SPN P ‐ and SPN C ‐injected and treated tumor‐bearing mice were unchanged relative to those in healthy mice (Figure 6C).…”

Recent Advances in Glycopeptide and Glycoprotein Synthesis: A Refined Synthetic Probe towards the Biological World

“…[87][88][89] In this regard, the chemical synthesis of an EPO glycoform bearing one triantennary (at Asn83) sialyl N-glycan was developed, in addition to the other two biantennary glycans (at Asn24 and Asn38, respectively). [90] The incorporation of the triantennary glycan relies on the isolation of Asn-linked biantennary nonasaccharide and conversion to its counterpart bearing triantennary undecasaccharide via selective chemical reactions (protection/deprotection and glycosylation); [88] subsequent solution phase coupling with two protected peptides, followed by global deprotection and enzymatic sialylation led to the assembly of EPO fragment (79)(80)(81)(82)(83)(84)(85)(86)(87)(88)(89)(90)(91)(92)(93)(94)(95)(96)(97); eventually the whole EPO molecule was obtained via sequential native chemical ligation, desulfurization and folding (Figure 14A). [90] Enhanced in vivo hematocrit activity was observed with the introduction of the additional trisaccharide at the biantennary glycan at Asn83 of EPO, compared with its biantennary counterpart.…”

“…Towards basically the same goal, the role of terminal sialic acid was probed via the development of another robust and reproducible chemoenzymatic approach, with the formation of glycoforms of EPO with and without sialic acids. [ 91 ] The three glycopeptides bearing biantennary N‐glycans with terminal galactose were obtained by pseudoproline‐assisted convergent aspartylation; the glycans were subsequently extended towards their sialylated forms, and the resulting glycopeptides were subjected to sequential native chemical ligation, desulfurization and folding (Figure 14B). While the enzymatic sialylation was readily accomplished at the level of the glycopeptide segments, the efficiency decreased with increasing chain length; impressively, the reaction proceeded even more efficiently on the refolded glycoprotein at multiple sites in a single step.…”

Enzymatic Sialylation of Synthetic Multivalent Scaffolds: From 3′‐Sialyllactose Glycomacromolecules to Novel Neoglycosides

“…[ 46 ] An alternative strategy relies on the use of enzymes to perform sialylation reactions. Inspired by the posttranslational modification of proteins, [ 47–49 ] where sialic acid residues are attached enzymatically. This is the last step of the synthetic procedure, [ 20,26,50 ] thereby also preventing degradation of the rather pH‐sensitive sialoside linkage and thus further improving yields.…”