Homogeneous Erythropoietin Research Articles

Recent Progress in the Synthesis of Homogeneous Erythropoietin (EPO) Glycoforms.

Erythropoietin (EPO) has been regarded as a therapeutic glycoprotein for the clinical treatment of anaemia since its approval by the Food and Drug Administration (FDA) in 1989. Commercial production of the 165-residue glycoprotein is by recombinant protein expression using mammalian cell lines that renders a complex mixture of glycoforms that have an identical amino acid sequence but variations in the structures of the pendant glycans. This heterogeneous nature of human recombinant EPO restricts structural and bioactivity studies in medicinal chemistry. Consequently, chemical synthesis provides an elegant approach for the preparation of complex homogeneous glycoproteins from a readily accessible pool of amino acids and sugars. In addition, the combination of chemical and biosynthesis enables robust and large-scale production of homogeneous EPO. The scope of this minireview is to summarise the recent advances in the chemical and semisyntheses of homogeneous EPO glycoforms, highlighting the versatile approaches to the preparation and structural manipulations of the carbohydrate chains incorporated into synthetic EPO glycoproteins.

Erythropoietin Derived by Chemical Synthesis

Erythropoietin is a signaling glycoprotein that controls the fundamental process of erythropoiesis, orchestrating the production and maintenance of red blood cells. As administrated clinically, erythropoietin has a polypeptide backbone with complex dishomogeneity in its carbohydrate domains. Here we describe the total synthesis of homogeneous erythropoietin with consensus carbohydrate domains incorporated at all of the native glycosylation sites. The oligosaccharide sectors were built by total synthesis and attached stereospecifically to peptidyl fragments of the wild-type primary sequence, themselves obtained by solid-phase peptide synthesis. The glycopeptidyl constructs were joined by chemical ligation, followed by metal-free dethiylation, and subsequently folded. This homogeneous erythropoietin glycosylated at the three wild-type aspartates with N-linked high-mannose sialic acid-containing oligosaccharides and O-linked glycophorin exhibits Procrit-level in vivo activity in mice.

ChemInform Abstract: The Winding Pathway to Erythropoietin Along the Chemistry—Biology Frontier: A Success at Last

AbstractReview: <70 refs.

The Winding Pathway to Erythropoietin Along the Chemistry–Biology Frontier: A Success At Last

The total synthesis of a homogeneous erythropoietin (EPO), possessing the native amino acid sequence and chitobiose glycans at each of the three wild-type sites of N glycosylation, has been accomplished in our laboratory. We provide herein an account of our decade-long research effort en route to this formidable target compound. The optimization of the synergy of the two bedrock sciences we now call biology and chemistry was central to the success of the synthesis of EPO.

At last: erythropoietin as a single glycoform.

At last: erythropoietin as a single glycoform.

Inside Cover: Chemical Synthesis of an Erythropoietin Glycoform Containing a Complex-type Disialyloligosaccharide (Angew. Chem. Int. Ed. 15/2012)

A homogeneous glycoprotein was prepared by the improved tert-Boc solid-phase peptide synthesis of acid-labile sialylglycopeptide α-thioesters, sophisticated peptide-coupling reactions, and native chemical ligation. In their Communication on page 3567 ff., Y. Kajihara and co-workers report the chemical synthesis of a homogeneous erythropoietin glycoform bearing a complex-type biantennary disialyloligosaccharide. This approach will open a new avenue for the investigation of oligosaccharide function.

Convergent chemical synthesis of [lysine(24,38,83)] human erythropoietin.

Erythropoietin (EPO) is a glycoprotein hormone that plays important roles in regulating the production of red blood cells (erythrocytes).[1] Since human EPO was first isolated and purified from urine in 1977,[2] the structure and physiological properties of EPO have been thoroughly studied.[3] Mature human EPO found in nature consists of a polypeptide chain of 165 amino acids with four covalently attached oligosaccharides,[4] one of which is an O-linked oligosaccharide at Ser126, and the other three are N-linked oligosaccharides at residues Asn24, 38, 83.[ 5 ] EPO is used as a therapeutic agent to treat anemia caused by chronic kidney disease.[6] Commercial EPO is prepared using recombinant DNA technology. The carbohydrate moieties of both native and recombinant EPO[7] are heterogenerous and composed of multiple glycans with different lengths and composition at each glycosylation site.[5a,8] This heterogeneity makes it difficult to evaluate the effects of carbohydrate on EPO’s pharmacokinetic properties and, more importantly, complicates the understanding of mechanism of EPO’s action at the molecular level. For these reasons, it is important to develop an alternative strategy to prepare homogeneous EPO.

Toward Homogeneous Erythropoietin: Application of Metal‐Free Dethiylation in the Chemical Synthesis of the Ala79‐Arg166 Glycopeptide Domain

We describe herein the assembly of hEPO(79-166), a key glycopeptide segment en route to erythropoietin, in minimally protected form. Key to the success of this synthetic endeavor was the application of our two-step cysteine-free native chemical ligation strategy, by which we achieved formal ligation at alanine and proline residues through the use of an N-terminal amino acid surrogate presenting a readily removable thiol functionality.

Design and Synthesis of a Homogeneous Erythropoietin Analogue with Two Human Complex‐Type Sialyloligosaccharides: Combined Use of Chemical and Bacterial Protein Expression Methods

Design and Synthesis of a Homogeneous Erythropoietin Analogue with Two Human Complex‐Type Sialyloligosaccharides: Combined Use of Chemical and Bacterial Protein Expression Methods

Inside Cover: Design and Synthesis of a Homogeneous Erythropoietin Analogue with Two Human Complex‐Type Sialyloligosaccharides: Combined Use of Chemical and Bacterial Protein Expression Methods (Angew. Chem. Int. Ed. 50/2009)

Inside Cover: Design and Synthesis of a Homogeneous Erythropoietin Analogue with Two Human Complex‐Type Sialyloligosaccharides: Combined Use of Chemical and Bacterial Protein Expression Methods (Angew. Chem. Int. Ed. 50/2009)

Design and Synthesis of a Homogeneous Erythropoietin Analogue with Two Human Complex‐Type Sialyloligosaccharides: Combined Use of Chemical and Bacterial Protein Expression Methods

Design and Synthesis of a Homogeneous Erythropoietin Analogue with Two Human Complex‐Type Sialyloligosaccharides: Combined Use of Chemical and Bacterial Protein Expression Methods

Toward homogeneous erythropoietin: chemical synthesis of the Ala1-Gly28 glycopeptide domain by "alanine" ligation.

The Ala(1)-Gly(28) glycopeptide fragment (28) of EPO was prepared by chemical synthesis as a single glycoform. Key steps in the synthesis include attachment of a complex dodecasaccharide (7) to a seven amino acid peptide via Lansbury aspartylation, native chemical ligation to join peptide 19 with the glycopeptide domain 18, and a selective desulfurization at the ligation site to reveal the natural Ala(19). This glycopeptide fragment (28) contains both the requisite N-linked dodecasaccharide and a C-terminal (alpha)thioester handle, the latter feature permitting direct coupling with a glycopeptide fragment bearing N-terminal Cys(29) without further functionalization.

Mature homogeneous erythropoietin building blocks by chemical synthesis: the EPO 22–37 glycopeptide domain presenting the full N-linked dodecasaccharide

A synthesis of EPO 22–37 glycopeptide ( 1), presenting the N-linked dodecasaccharide of erythropoietin, is described.