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.

ICP-OES was measured on an Optima 7300 DV from PerkinElmer with a Meinhard atomizer.
CMP-Neu5Ac was kindly provided by Roche Diagnostics GmbH as sodium salt.
α-2,6-Sialyltransferase from Photobacterium damsela (Δ15PD2,6ST) [3] was obtained from Sigma Aldrich. Alkaline phosphatase from calf intestine was purchased from Sigma Aldrich The degree of loading of resins was determined by Fmoc cleavage of the penultimate amino acid. [4,5] Absorbance measurements were performed on a Specord 2000 spectrophotometer from Analytik Jena. Analytical TFA deprotections were performed by adding 0.5 mg of resin or 0.2 mg of protected peptide to 100 μL of TFA/TIS/H2O (95:2.5:2.5) for 60 min. The mixture was dried in high vacuum, the residue was dissolved in MeCN/H2O + 0.1 % HCOOH and analyzed by HPLC-MS.

Automated Fmoc-SPPS
Solid-phase synthesis following the Fmoc strategy [6] was performed automatically in 45 mL reaction vessels on a Tribute peptide synthesizer with IntelliSynth UV Monitoring and Feedback Control System from Protein Technologies Inc. All reactions were performed at ambient temperature under a nitrogen atmosphere and mechanical shaking. Nitrogen was passed through the reaction suspension for additional mixing. Before starting the synthesis, the resin was swollen by washing with CH2Cl2 (5 x 30 s) and DMF (5 x 30 s).

Automated Removal of N α -Fmoc
To remove N-terminal Fmoc protection a piperidine solution (20 % in DMF) was added to the resin and shaken for 0.5 min. The coupled UV monitoring system of the peptide synthesizer initiated further Fmoc cleavage cycles if the absorbance (A301) of the filtrate indicated incomplete cleavage. After complete Fmoc removal, the resin was washed with DMF (5 x 30 s).

Automated Coupling of Fmoc amino acid derivatives
The amino acid building block and the activation reagent were taken up in a defined volume of DIPEA in DMF (for molarity see specific synthesis) before coupling, mixed for 2 min, and added to the deprotected peptidyl resin. Cysteine derivatives were activated as symmetric anhydrides and coupled manually. At the end of each coupling the resin was washed with DMF (5 x 30 s). The exact coupling conditions are given in the specific synthesis protocols.
Manual coupling of cysteine derivatives [7] In dried glassware under argon, the cysteine building block (10 equiv.) was dissolved in dry DMF/CH2Cl2 1:2.5 (c Fmoc/Boc-Cys(PG)-OH = 180 mM) and N,N'-diisopropylcarbodiimide (5 equiv.) was added at 0 °C. The reaction mixture was stirred at 0 °C for 5 min and for 30 min at ambient temperature. Subsequently, the CH2Cl2 was removed under vacuum. The suspension was drawn up into a syringe with and added to the deprotected peptidyl resin swollen in DMF.
The residue and the vessel were washed three times with an appropriate volume of DMF to give a concentration of 105 mM for the anhydride in the coupling solution. After 3 -5 h of shaking at ambient temperature, the coupling solution was removed and the resin was washed with CH2Cl2 and DMF (five times each).

Mild acidic cleavage of protected peptidyl acids from trityl resin
To cleave protected peptidyl acids from p-carboxytrityl-ChemMatrix resin the peptidyl resin was swelled in CH2Cl2 in a syringe reactor for 20 min. Subsequently 20 % HFIP in CH2Cl2 (approx. 8 mL/g resin) was added and the suspension was shaken at room temperature for 1 min.
The resin was treated seven times for 1 min each with the same volume of 20 % HFIP solution and finally washed five more times with CH2Cl2. All the cleavage and washing solutions were collected in a cooled flask (0 °C) containing 25 times the volume of CH2Cl2 used for a single cleavage. The combined cleavage solutions were concentrated under vacuum at 4 °C bath temperature. HFIP was azeotropically removed by adding CH2Cl2 several times. The residue was dried in vacuo and lyophilized from dioxane.
Subsequently, 0.5 M NaNO2 (6.5 eq) was added under stirring. After 1 h at -15 °C MESNa (65 equiv., 1.0 M in 6 M GdmCl/0.2 M NaH2PO4) was added. The pH value of the MESNa solutions was set to 6.6 for EPO 29-67 glycopeptides 21 and 35 and to pH 6.0 for EPO 29-97 glycopeptides S14 and S15. The concentration of the acyl azide in the thiolysis mixture was 2.75 mM. Once the reaction mixture reached ambient temperature a microelectrode was used to readjust the pH using 6 M and 1 M NaOH. At the desired pH the reaction mixture was stirred for 1 h with the microelectrode immersed. Subsequently, the microelectrode and the magnetic stir bar were rinsed with 100 μL of phosphate buffer (0.2 M NaH2PO4/6 M GdmCl/pH 3.0). For sialylated compounds 0.2 M NaH2PO4/6 M GdmCl/pH 6.6 (EPO 29-67 glycopeptide 35) or pH 6.0 (EPO29-97 glycopeptide S15) were used in the rinsing step. The combined reaction mixtures were desalted by gel filtration.
351.4 mg (130 µmol, 1 eq) of resin S10 were placed in a 45 mL peptide synthesizer reaction vessel and the peptide chain was elongated automatically. For cleavage of the Fmoc group the general procedure was applied and the amino acid building blocks were coupled under the conditions listed in table S3 and the general procedure section. After complete elongation the resin S11 was washed with DMF (5x) and CH2Cl2 (5x) and was dried in vacuo.
300.0 mg (75 µmol, 1 eq) of resin S12 were placed in a 45 mL peptide synthesizer reaction vessel and the peptide chain was elongated automatically. For cleavage of the Fmoc group the general procedure was applied and the amino acid building blocks were coupled under the conditions denoted in table S4 and the general procedure section. After complete elongation the resin S13 was washed with DMF (5 x) and CH2Cl2 (5 x
According to the general procedure for thioester formation, the glycopeptide hydrazide 20         Figure S22: Synthesis of EPO 29-97 thioester S14

Synthesis of EPO 1-28 Sialoglycopeptide Thioester 34
According to the general procedure for thioester formation, the sialoglycopeptide hydrazide 31   According to the general procedure for thioester formation, the sialoglycopeptide hydrazide 32     Figure S48: Synthesis of EPO 29-97 glycopeptide thioester S15.