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

Department of Chemistry, Graduate School of Science, Osaka University, 1-1, Machikaneyama, Toyonaka, 560-0043 Japan.
Angewandte Chemie International Edition (Impact Factor: 11.26). 04/2012; 51(15):3567-72. DOI: 10.1002/anie.201109034
Source: PubMed


New and improved: New reaction conditions for tert-Boc-based solid-phase peptide synthesis make acid-labile sialyloligosaccharyl peptide α-thioesters accessible. To demonstrate this, a sialyloligosaccharyl-erythropoietin glycoform with 166 amino acid residues was synthesized.

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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 a and b subunits (Aussedat et al., 2012; Nagorny et al., 2012), glycosylated human interferon-b (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 thioester 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). "
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    ABSTRACT: 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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    ABSTRACT: Biosynthesis of glycoproteins in the endoplasmic reticulum employs a quality control system, which discriminates and excludes misfolded malfunctional glycoproteins from a correctly folded one. As chemical tools to study the glycoprotein quality control system, we systematically synthesized misfolded homogeneous glycoproteins bearing a high-mannose type oligosaccharide via oxidative misfolding of a chemically synthesized homogeneous glycopeptide. The endoplasmic reticulum folding sensor enzyme, UDP-glucose:glycoprotein glucosyltransferase (UGGT), recognizes a specific folding intermediate, which exhibits a molten globule-like hydrophobic nature.
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    ABSTRACT: High-mannose type oligosaccharides consist of nine mannose and two N-acetylglucosamine residues (Man(9)GlcNAc(2):M9) and play an important role in protein folding processes in the endoplasmic reticulum. A highly efficient preparation method of this asparaginyl-M9-oligosaccharide from hen egg yolk was established by a two-step proteolysis with commercially available proteases and subsequent purification using high performance liquid chromatography (HPLC). To avoid the hydrolysis of the desired M9-oligosaccharide during the proteolysis steps, several commercially available proteases were screened for their contamination with mannosidases. The α-amino group of the resultant H(2)N-Asn-(M9-oligosaccharide)-OH was protected with 9-fluorenylmethyloxycarbonyl (Fmoc) group for convenient separation by HPLC. The structure of Fmoc-Asn-(M9-oligosaccharide)-OH thus obtained was confirmed by ESI-MS spectrometry and several NMR experiments. Using this Fmoc-Asn-(M9-oligosaccharide)-OH, the synthesis of the M9-glycopeptide-α-thioester was demonstrated by means of tert-Boc-solid phase peptide synthesis. These tert-Boc conditions afforded the M9-glycopeptide-α-thioester in moderate yield.
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