A synthetic approach to 'click' neoglycoprotein analogues of EPO employing one-pot native chemical ligation and CuAAC chemistry

Abstract

The clinical significance of batch-wise variability on the pharmacokinetics and potency of commercial erythropoietin (EPO), prepared recombinantly as a heterogeneous mixture of glycoforms, necessitates the development of synthetic strategies to afford homogenous EPO formulations. Herein we present a previously unexplored and divergent route towards 'click' neoglycoprotein analogues of EPO, employing one-pot native chemical ligation (NCL) of alkynylated peptides and copper-catalysed azide-alkyne cycloaddition (CuAAC) with azido monosaccharides. By design, our synthetic platform permits glycosylation at virtually any stage, providing flexibility for the synthesis of various glycoforms for biological analysis. Insights obtained from attempted folding of our 'click' neoglycoprotein EPO analogue, bearing four different neutral sugar moieties, highlight the important role played by the charged oligosaccharides present in native EPO glycoproteins.

Fig. 1. (a) One-pot synthesis of ‘click’ neoglycopeptides employing CuAAC ‘click’ chemistry and native chemical ligation (NCL); (b) specific amino acid (2 and 3) and azido-monosaccharide (4–7) building blocks employed in this work; (c) polypeptide sequence of des-Arg¹⁶⁶ human erythropoietin (1), divided into 5 peptide fragments for convergent synthesis and highlighting the location of relevant residues: X = click neoglycosylation sites = propargylglycine = Pra; C= Cys(Acm), Acm = acetamidomethyl; Z = Thz = l-thiazolidine-4-carboxo; W = Trp(CHO).

Scheme 1. Initial synthetic strategy towards a ‘click’ neoglyco-EPO analogue. For graphical simplicity, GalNAc-N₃ (6) is shown as the sugar component; other glycans can be substituted in a straightforward manner.

Fig. 2. (a) Initial synthetic approach towards ‘click’ EPO3-5 neoglycopeptide 15, employing one-pot NCL and CuAAC chemistry. Reagents and conditions: NCL: MPAA (20 mM), TCEP·HCl (40 mM), 6 M GnHCl/0.2 M Na₂HPO₄, pH 6.8, 7 h; CuAAC: CuSO₄ (20 mM), TCEP·HCl (10 mM), GalNAcα1-O-(CH₂)₃N₃7 in the same pot, 50 °C; (b–f) analytical LC-MS traces for the one-pot NCL and click reaction between EPO4-5 (14), EPO3 (10) peptides and GalNAcα1-O-(CH₂)₃N₃7; (b) t = 1 h; (c) t = 7 h. The ligated Thz-EPO3-5 ligation peptide was observed in near quantitative yield; (d) t= 5 h after start of click reaction. The bisglycosylated ‘click’-EPO3-5 neoglycopeptide product (15) was obtained in high purity; (e) purified ‘click’-EPO3-5 (15); (f) mass spectrum of ‘click’-EPO3-5 (15) (observed: [M + 6H]⁶⁺ = 1903.22 Da, calculated: 1903.52 Da). Analytical monitoring was carried out using a Phenomenex Gemini C₁₈ column (110 Å, 50 mm × 2.0 mm; 5 μm) using a gradient of 5–65% buffer B over 30 min (buffer A= 0.1% TFA in H₂O; buffer B= 0.1% TFA in acetonitrile) at 210 nm.

Fig. 3. (a) Alternative synthetic approach towards ‘click’ EPO3-5 neoglycopeptide 16, bearing two different ‘click’ neoglycans. Reagents and conditions: NCL between EPO4 (11) and EPO5 (12): 6 M GnHCl/0.2 M Na₂HPO₄, MPAA (200 mM), TCEP·HCl (50 mM) pH 6.8, 2.5 h; SPE using C4 semi-prep column and lyophilization, then CuAAC with sugar azide 7: 6 M GnHCl/0.2 M Na₂HPO_4, GalNAcα1-O-(CH₂)₃–N₃ (7) (5 mM), CuSO₄ (40 mM) and TCEP·HCl (20 mM), 40 °C, pH 7, 2 h. NCL between ‘click’ EPO4-5 (17) and EPO3 (10): 6 M GnHCl/0.2 M Na₂HPO₄, MPAA (100 mM), TCEP·HCl (40 mM), pH 6.8, 2 h; SPE using C4 semi-prep column and lyophilization, then CuAAC with sugar azide 5: 6 M GnHCl/0.2 M Na₂HPO_4, Glc-N₃5 (5 mM), CuSO₄ (40 mM) and TCEP·HCl (20 mM), 50 °C, pH 7, 4 h; (b) analytical LC monitoring of the NCL-CuAAC sequence between EPO4 (11), EPO5 (12) and sugar azide (7). Mass spectrum of pure ‘click’ EPO4-5 (17) (observed: [M + 5H]⁵⁺ = 1555.78 Da, calculated: 1555.99 Da). The analytical monitoring was carried out using an analytical column (Phenomenex Jupiter C4, 300 Å, 50 mm × 2.0 mm; 5 μm) using a gradient of 5–65% buffer B over 30 min (buffer A= 0.1% TFA in H₂O; buffer B= 0.1% TFA in acetonitrile) at 210 nm; (c) analytical LC monitoring of the NCL-CuAAC sequence between EPO3 (10), ‘click’ EPO4-5 (17) and sugar azide 5. Mass spectrum of pure ‘click’ EPO3-5 neoglycopeptide product (16) (observed: [M + 8H]⁸⁺ = 1415.46 Da, calculated: 1415.50 Da). The analytical monitoring was carried out using an analytical column (Phenomenex Jupiter C4, 300 Å, 50 mm × 2.0 mm; 5 μm) using a gradient of 5–65% buffer B over 30 min (buffer A= 0.1% TFA in H₂O; buffer B= 0.1% TFA in acetonitrile) at 210 nm.

Scheme 2. Revised route to ‘click’ EPO1-2 (21). The initial route (Scheme 1) failed due to the inefficiency of KCL between EPO1-MPAA and EPO2 peptides. The revised strategy involved a one-pot CuAAC and MPAA thioester exchange reaction on EPO1 peptide 8, followed by one-pot KCL and click chemistry with the EPO2 peptide 9 using a different sugar. This route allows for diversity in sugars to be attached to the resulting ‘click’ EPO1-2 neoglycopeptide fragment 21.

Fig. 4. (a) ‘One-pot’ synthetic approach to ‘click’ EPO1-2 neoglycopeptide 21, bearing two different ‘click’ neoglycans. Reagents and conditions: 6 M GnHCl/0.2 M Na₂HPO₄, pH 6.3, 6 h, then MESNa (100 mM); Gal-N₃4 (5 mM), CuSO₄ (20 mM) and TCEP·HCl (10 mM), added to the same pot, r.t., 5 h; (b) analytical LC monitoring of the one-pot KCL-CuAAC sequence between ‘click’ EPO1-MPAA (20), EPO2 (9) and sugar azide 4; (c) mass spectrum of ‘click’ EPO1-2 neoglycopeptide 21 (observed: [M + 4H]²⁺ = 2127.63 Da, calculated: 2128.40 Da). The analytical monitoring was carried out using a Phenomenex Jupiter C4 column (300 Å, 50 mm × 2.0 mm; 5 μm) using a gradient of 5–65% buffer B over 30 min (buffer A= 0.1% TFA in H₂O; buffer B= 0.1% TFA in acetonitrile) at 210 nm.

Fig. 5. (a) NCL and deformylation to access ‘click’ EPO1-5 neoglycopeptide 22 bearing four different ‘click’ neoglycans. Reagents and conditions: NCL and deformylation) 6 M GnHCl/0.2 M Na₂HPO₄, MPAA (200 mM), TCEP·HCl (50 mM), pH 6.8, 3 h; then β-mercaptoethanol (400 μL) and piperidine (250 μL), 10 min; SPE using C4 semi-prep column and lyophilization; (b–d) analytical LC monitoring of the ligation between ‘click’ EPO1-2 (21) and ‘click’ EPO3-5 (16); (b) t = 10 min of NCL; (c) t = 3 h, the NCL reaction was essentially complete to yield the ligation product ‘click’ EPO1-5 [Cys(Acm)^7,29,33,161][Cys^30,68,98,128][Trp(CHO)^51,64,88]; (d) t = 10 min of deformylation to yield ‘click’ EPO1-5 [Cys(Acm)^7,29,33,161][Cys^30,68,98,128] (22); (e) mass spectrum of ‘click’ EPO1-5 (22) purified by SPE only (observed: [M + 14H]¹⁴⁺ = 1398.39 Da, calculated: 1398.45 Da. Deconvoluted mass observed: 19 564.63 Da, calculated: 19 564.34 Da). The analytical monitoring was carried out using an analytical column (Phenomenex Jupiter C4, 300 Å, 50 mm × 2.0 mm; 5 μm) employing a gradient of 5–65% buffer B over 30 min (buffer A = 0.1% TFA in H₂O; buffer B = 0.1% TFA in acetonitrile) at 210 nm.

Fig. 6. (a) Graphical representation of attempted disulfide formation and protein refolding from the unfolded ‘click’ EPO1-5 neoglycoprotein 24; (b and c) analytical RP-HPLC chromatograms employing an analytical column (Phenomenex Jupiter C4, 300 Å, 50 mm × 2.0 mm; 5 μm) and a gradient of 5–65% buffer B over 30 min (buffer A= 0.1% TFA in H₂O; buffer B= 0.1% TFA in acetonitrile); (b) RP-HPLC of pure ‘click’ EPO1-5[Cys^7,29,33,161][Ala^30,68,98,128] neoglycoprotein 24; (c) analytical LC monitoring of the refolding reaction at 210 nm. Upper spectra: t = 0 h, Lower spectra: t = 19 h; (d) mass spectrum after 19 h of folding. The left of the two peaks corresponded to the correctly folded protein ‘click’ EPO (25) (deconvoluted mass: 19 147.68 Da, calculated: 19 147.77 Da). The right of the two peaks in the mass spectrum corresponded to the partially folded protein adduct.