introduction
Erythropoietin (EPO) is a hormone that plays important roles in erythropoiesis. The sequence of EPO consists of 166 residues, including three N-glycosylation sites at Asn24, 38 and 83. Although EPO perform important biofunction as a glycoprotein, the function of its N-glycans still not fully understood. The synthesis of homogeneous N-glycosylated EPO for elucidation of its N-glycan function was studied by chemists. In 2012, Murakami and Kajihara group achieved the total synthesis of folded erythropoietin bearing a complex type sialyloligosaccharide at Asn83 (Fig6: Synthetic strategy of the total synthesis of erythropoietin by efficient phenacyl protection).[25] The traditional Boc-SPPS method was challenging for synthesizing disialo-glycopeptide thioester due to the acidity-labile feature of sialic acid residue. To solve this problem, a synthetic strategy was developed based on phenacyl (Pac)-protection of sialic acid on N-glycan. After coupling of glycan on resin, the protected disialo N-glycan keep intact under acidic condition for both t-Boc deprotection (TFA) and global deprotection (TFA/TfOH/DMS/m-cresol). Finally, to test the practicability of this method, glycoprotein EPO having disialo N-glycan at Asn83 was chemically synthesized. Four segments 56, 57, 58 and 59 were ligated sequentially by NCL. The glycopeptide (sequence:50-166) was subjected to afford desulfurized glycopeptide 60 of the three free thiol groups. A final ligation and deprotection of Acm protecting group of four native cysteines gave the liner EPO. Following oxidative folding protocols afforded an EPO 61 with one disialyloligosaccharide. CD spectra and MS spectra suggested correct folding.
Then in 2016, Murakami and Kajihara group succeeded in the synthesis of glycoproteins EPO of which N-glycosylation numbers and positions were varied from one to three.[26] In this synthesis, they installed complex type biantennary sialylglycan at Asn24, Asn38, Asn83 and as a result they synthesized five EPO glycoforms varying in glycosylation positions and the numbers of human-type biantennary sialyloligosaccharides (Fig7: Total synthesis of five glycoforms of EPO varying in glycosylation positions and the numbers of human-type biantennary sialyloligosaccharides). Furthermore, they discussed structure and activity relationship. Consequently, this example became the first work to give an insight into the relationship between glycosylation pattern and hematopoietic activity. In addition to this synthesis, based on the same assemble strategy, Maki and Kajihara achieved the installation complex type triantennary sialylglycan at Asn83 and as a result they synthesized alternative EPO glycoforms with triantennary sialyloligosaccharides 74.[21]
