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. The in-vivo activity of EPO is carbohydrate-dependent with the number of sialic acid residues regulating its circulatory half-life. EPO carries three N-glycans and thus obtaining pure glycoforms provides a major challenge. We have developed a robust and reproducible chemoenzymatic approach to glycoforms of EPO with and without sialic acids. EPO was assembled by sequential native chemical ligation of two peptide and three glycopeptide segments. The glycopeptides were obtained by pseudoproline-assisted 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 1:1 complexes with recombinant EPO receptor.

Keywords: glycopeptides; glycoproteins; native chemical ligation; oligosaccharide; receptor.

Scheme 1

Retrosynthesis of human EPO 1–166 with three asialo N‐glycans leading to fragments 1–5.

Scheme 2

Drawbacks of the initial protecting group strategies for native cysteines: a) loss of Acm during Pd⁰‐catalyzed deallylation; b) N‐terminal Thz forming a stable nitroso derivative under diazotization conditions; c) difficult removal of Phacm at a late stage using various deprotection methods; d) low overall yield for S‐tritylation and low solubility in following transformations.

Scheme 3

Solid phase synthesis and functionalization of the five EPO segments. The glycopeptide hydrazides 17, 20 and 23 were obtained using pseudoproline‐assisted Lansbury aspartylation.

Scheme 4

a) Synthesis of the asialoglycoform EPO A bearing three N‐glycans by sequential native chemical ligation, desulfurization, Pd‐mediated cleavage of the Acm groups and oxidative refolding; b) RP‐HPLC‐MS of the final ligation of glycopeptide thioester 18 and glycopeptide 29 to full‐length EPO 1–166 30 after 6 d, insert shows SDS‐PAGE of crude ligation mixture; c) SEC after oxidative refolding of ligation mixture; d) RP‐HPLC‐MS of EPO A after RP‐HPLC purification; e) HR‐MS of purified EPO A (H₂O, direct injection); f) simulated and measured isotope pattern of the [M+15 H]¹⁵⁺ HR‐MS peak; g) SDS‐PAGE (reduced and oxidized) of EPO A.

Scheme 5

The efficiency of the enzymatic 2,6‐sialylation of EPO glycopeptides decreases with increasing chain length: a) sialylation of 30–40mer glycopeptide hydrazides 17, 20, 23 and RP‐HPLC‐MS of crude mixtures; b) sialylation of EPO 29–97 glycopeptide 25 and RP‐HPLC‐MS of crude mixtures; c) sialylation of EPO 29–166 glycopeptide 28; Total Ion Chromatogram (TIC) of reaction mixture RP‐HPLC‐HRMS after 31 h; RP‐HPLC‐HRMS of reaction mixtures after different reaction times (only 11+ charged peaks of MS are shown).

Scheme 6

a) Synthesis of glycoform EPO S from sialylated glycopeptides; b) RP‐HPLC‐MS of the ligation of sialylated glycopeptide thioester 34 and Cys‐glycopeptide 29–166 S 16 to full‐length EPO 1–166 39 after 6 d, c) SEC after oxidative refolding; d) RP‐HPLC‐MS of EPO S after RP‐HPLC purification; e) HR‐MS of purified EPO S (H₂O, direct injection); f) simulated and measured isotope pattern of [M+15 H]¹⁵⁺ HR‐MS peak; g) SDS‐PAGE (reduced and oxidized) of EPO S. h) CD‐spectra of EPO A and EPO S.

Scheme 7

a) The enzymatic sialylation of EPO A yields the sialylated glycoform EPO S directly; b) RP‐HPLC‐HRMS of the enzymatic sialylation of EPO A after 22 h, sialylation intermediates coelute with product EPO S: TIC of LC‐MS (1.5 h) and MS after different reaction times are shown; c) RP‐HPLC‐MS of EPO S after RP‐HPLC purification; d) HR‐MS of purified EPO S (H₂O, direct injection); e) simulated and measured isotope pattern of [M+15 H]¹⁵⁺ HR‐MS peak; f) SDS‐PAGE of EPO S (reduced and oxidized).

Scheme 8

a) Cartoon of the high affinity (1:1) complexes of EPO A and EPO S with EPOR based on a crystal structure (PDB code: 1EER); b) complex formation of EPO A, EPO S and EPO_CHO with EPOR monitored by SEC (apparent molecular weights of the complexes were calculated by linear approximation between the reference points of 75 and 44 kDa given by the manufacturer); c) SDS‐PAGE (non‐reducing) of the complexes of EPO A, EPO S and EPO_CHO with EPOR isolated by SEC; d) the isolated complexes of EPO A, EPO S and EPO_CHO with EPOR were first dissociated with 0.2 % TFA and subsequently analyzed by RP‐HPLC‐MS.