Chemical Synthesis of an Erythropoietin Glycoform Containing a Complex‐type Disialyloligosaccharide

Murakami, Masumi; Okamoto, Ryo; Izumi, Masayuki; Kajihara, Yasuhiro

doi:10.1002/ange.201109034

Cited by 64 publications

(40 citation statements)

References 70 publications

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“…This development has provided the flexibility to retrosynthetically disconnect the glycoprotein sequence at appropriate junctions and significantly expanded the scope of NCL for protein and glycoprotein synthesis (Payne and Wong, 2010; Unverzagt and Kajihara, 2013). The recent success in the chemical synthesis of glycoprotein hormone α- and β-subunits (Aussedat et al, 2012; Nagorny et al, 2012), glycosylated human interferon-β (Sakamoto et al, 2012), and full-size erythropoietin (Murakami et al, 2012; Wang et al, 2012) showcases the power of the ligation methods for total glycoprotein synthesis. Moreover, expressed protein ligation (EPL) has been successfully explored for glycoprotein synthesis, in which a large intact protein thiosester or an N-terminal Cys-containing protein domain is recombinantly expressed and used as ligation partners (Muir, 2003; Muir et al, 1998; Payne and Wong, 2010; Schwarzer and Cole, 2005).…”

Section: Major Approaches For Glycoprotein Synthesismentioning

confidence: 99%

“…The relatively large size and complex glycosylation pattern of EPO make it an attractive and challenging synthetic target for chemists to test new synthetic strategies (Wilson et al, 2013). Kajihara and co-workers recently reported a total chemical synthesis of a full-length EPO glycoform that carries a sialylated bi-antennary complex N-glycan at the Asn-83 site (Murakami et al, 2012). Instead of using the existing 4 Cys residues for NCL, which are unfavorably positioned close to the N or C-terminus of EPO, the authors decided to disconnect the EPO sequence at 5 appropriate Ala sites to provide 5 peptide fragments and 1 glycopeptide (aa 79–97) fragment, which can be readily synthesized by SPPS (Figure 3a).…”

Section: Total Chemical Synthesis Of Erythropoietin (Epo)mentioning

confidence: 99%

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Chemical and Chemoenzymatic Synthesis of Glycoproteins for Deciphering Functions

Wang

Amin

2014

Chemistry & Biology

149

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Summary Glycoproteins are an important class of biomolecules involved in a number of biological recognition processes. However, natural and recombinant glycoproteins are usually produced as mixtures of glycoforms that differ in the structures of the pendent glycans, which are difficult to separate in pure glycoforms. As a result, synthetic homogeneous glycopeptides and glycoproteins have become indispensable probes for detailed structural and functional studies. A number of elegant chemical and biological strategies have been developed for synthetic construction of tailor-made, full-size glycoproteins to address specific biological problems. In this review, we highlight recent advances in chemical and chemoenzymatic synthesis of homogeneous glycoproteins. Selected examples are given to demonstrate the applications of tailor-made, glycan-defined glycoproteins for deciphering glycosylation functions.

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Section: Major Approaches For Glycoprotein Synthesismentioning

confidence: 99%

Section: Total Chemical Synthesis Of Erythropoietin (Epo)mentioning

confidence: 99%

Chemical and Chemoenzymatic Synthesis of Glycoproteins for Deciphering Functions

Wang

Amin

2014

Chemistry & Biology

149

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“…[13] Conventional Boc SPPS cannot be used for the synthesis of peptides containing a complex glycan moiety because of the instability of the glycan moiety in strong acid media particularly the anhydrous HF used in the final deprotection step. [18] Thus, Fmoc (9-fluorenylmethoxycarbonyl) SPPS, which is compatible with glycopeptide synthesis, is used to make such glycopeptides. However, the synthesis of peptidea thioesters is not compatible with Fmoc SPPS because the thioester moiety is not stable to the repeated treatments with concentrated piperidine solution used to remove the Na Fmoc group at each stage of the synthesis.…”

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confidence: 99%

Total Chemical Synthesis and Biological Activities of Glycosylated and Non‐Glycosylated Forms of the Chemokines CCL1 and Ser‐CCL1

et al. 2014

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CCL1 is a naturally glycosylated chemokine protein that is secreted by activated T-cells and acts as a chemoattractant for monocytes.[1] Originally, CCL1 was identified as a 73 amino acid protein having one N-glycosylation site, [1] and a variant 74 residue non-glycosylated form, Ser-CCL1, has also been described. [2] There are no systematic studies of the effect of glycosylation on the biological activities of either CCL1 or Ser-CCL1. Here we report the total chemical syntheses of both N-glycosylated and non-glycosylated forms of (Ser-)CCL1, by convergent native chemical ligation. We used an N-glycan isolated from hen egg yolk together with the Nbz linker for Fmoc chemistry solid phase synthesis of the glycopeptidea thioester building block.[3] Chemotaxis assays of these glycoproteins and the corresponding non-glycosylated proteins were carried out. The results were correlated with the chemical structures of the (glyco)protein molecules. To the best of our knowledge, these are the first investigations of the effect of glycosylation on the chemotactic activity of the chemokine (Ser-)CCL1 using homogeneous N-glycosylated protein molecules of defined covalent structure.

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“…[4] Der Zugang zu einer reinen Glycoform von EPO mit allen vier Glycanen war bislang verschlossen. [1] Die Glycosylierung von Proteinen ist an verschiedensten Prozessen beteiligt, einschließlich Befruchtung, Immunüberwachung, Hormonaktivität, neuronale Entwicklung und Proteinfaltung.…”

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“…[4] Der Zugang zu einer reinen Glycoform von EPO mit allen vier Glycanen war bislang verschlossen. Es gibt eine Reihe eleganter Synthesen von EPO-Analoga.…”

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Totalsynthese von Erythropoietin: ein Ergebnis der Entwicklung wegbereitender Synthesetechniken

Payne

2012

Angewandte Chemie

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Chemische Ligation · Glycopeptide · Glycoproteine · Glycosylierungen · Totalsynthese Glycosylierungen sind die häufigsten posttranslationalen Modifikationen bei Proteinen, und geschätzte 50-70 % aller Proteine des Menschen tragen kovalent gebundene Glycane. [1] Die Glycosylierung von Proteinen ist an verschiedensten Prozessen beteiligt, einschließlich Befruchtung, Immunüberwachung, Hormonaktivität, neuronale Entwicklung und Proteinfaltung. [2] Trotz der unbestreitbaren Bedeutung der Glycosylierung war es bisher mühsam, die Auswirkungen dieser Modifikation auf die Struktur und Funktion von Proteinen aufzuklären. Einer der Hauptgründe hierfür ist, dass solche Glycoproteine in reiner Form schwer zugänglich sind. Dieses Problem entsteht insbesondere dadurch, dass, anders als die Proteinsynthese, der Glycosylierungsprozess nicht an einer Matrize stattfindet. Glycosylierungen werden vielmehr von Glycosyltransferasen gesteuert, die zu Mischungen von Glycoformen führen. Da sich diese Glycoformen nur in der Art der kovalent gebundenen Glycane unterscheiden, sind sie mit chromatographischen Methoden oftmals unmçglich zu trennen. Rekombinantes humanes Erythropoietin (rhEPO), ein Multimilliarden-Dollar-Wirkstoff zur Behandlung von Anämie, ist ein aus 166 Aminosäuren aufgebautes Protein mit vier Glycosylierungsstellen: drei hoch variable N-verknüpfte Stellen (an Asn-24, Asn-38 und Asn-83) und eine stärker konservierte O-verknüpfte Stelle (an Ser-126). Als Folge seiner Produktion in Säugerzellen wird rhEPO als komplexe Mischung von Glycoformen vertrieben, und es ist unbekannt, welcher der Glycosylierungszustände von EPO die hçchste Aktivität hat.Wildtyp-EPO, in dem alle vier Glycosylierungsstellen mit Glycanen besetzt sind, ist das ultimative Ziel aller Synthesebemühungen und war daher Gegenstand intensiver Forschungen. Es gibt eine Reihe eleganter Synthesen von EPO-Analoga. [3] Kürzlich beschrieb die Arbeitsgruppe von Kajihara die Synthese von Volllängen-EPO [allerdings mit zwei Aminosäuremutationen (Glu21Ala and Gln78Ala) zur Erleichterung der Synthese] mit einem komplexen sialylierten biantennären Glycan. [4] Der Zugang zu einer reinen Glycoform von EPO mit allen vier Glycanen war bislang verschlossen.

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Chemical Synthesis of an Erythropoietin Glycoform Containing a Complex‐type Disialyloligosaccharide

Cited by 64 publications

References 70 publications

Chemical and Chemoenzymatic Synthesis of Glycoproteins for Deciphering Functions

Chemical and Chemoenzymatic Synthesis of Glycoproteins for Deciphering Functions

Total Chemical Synthesis and Biological Activities of Glycosylated and Non‐Glycosylated Forms of the Chemokines CCL1 and Ser‐CCL1

Totalsynthese von Erythropoietin: ein Ergebnis der Entwicklung wegbereitender Synthesetechniken

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