Erythropoietin derived by chemical synthesis

Abstract

Erythropoietin is a signaling glycoprotein that controls the fundamental process of erythropoiesis, orchestrating the production and maintenance of red blood cells. As administrated clinically, erythropoietin has a polypeptide backbone with complex dishomogeneity in its carbohydrate domains. Here we describe the total synthesis of homogeneous erythropoietin with consensus carbohydrate domains incorporated at all of the native glycosylation sites. The oligosaccharide sectors were built by total synthesis and attached stereospecifically to peptidyl fragments of the wild-type primary sequence, themselves obtained by solid-phase peptide synthesis. The glycopeptidyl constructs were joined by chemical ligation, followed by metal-free dethiylation, and subsequently folded. This homogeneous erythropoietin glycosylated at the three wild-type aspartates with N-linked high-mannose sialic acid-containing oligosaccharides and O-linked glycophorin exhibits Procrit-level in vivo activity in mice.

Fig. 1. Schematic representation of the target homogenous EPO glycoform 1

Fig. 2. Synthesis of revised glycopeptide fragments

Reaction conditions for Sakakibaraelongation(26):Peptidylacid, amino acidthioester, EDC free base, HOOBt, CHCl₃/TFE, room temperature (rt). Reaction conditions for aspartylation: (i) Peptidyl thioester, S2, HATU, DIEA, DMSO, rt. (ii) TFA/TIS/H₂O/phenol, rt. EDC = N-(3-Dimethylaminopropyl)- N’-ethylcarbodiimide; HOOBt = 3-Hydroxy-1,2,3-benzotriazin-4(3H)-one; HATU = 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; TFE = trifluoroethanol; DIEA = N,N-Diisopropylethylamine; DMSO = dimethyl sulfoxide; TFA = trifluoroacetic acid; TIS = triisopropylsilane.

Fig. 3. Synthesis of homogeneous EPO glycoform 1

SPPS, solid-phase peptide synthesis. Reaction conditions for NCL: 6 M GND•HCl, 300 mM Na₂HPO₄, 20 mM TCEP•HCl, 200 mM MPAA, pH 7.2, rt. Reaction conditions for Thz opening: MeONH₂•HCl, rt. Reaction conditions for MFD: 5.7 M GND•HCl, 200 mM Na₂HPO₄, 300 mM TCEP (Bond-Breaker), t-BuSH, VA-044, pH 6.8, 37°C. Reaction conditions for Acm removal: AgOAc, AcOH/H₂O, rt. Reaction conditions for folding: cysteine/cystine (8:1). GND = guanidine; TCEP = tris(2-carboxyethyl)phosphine; MPAA = 4-Mercaptophenylacetic acid; Thz = thiazolidine; VA-044 = 2,2′-Azobis[2-(2-imidazolin-2-yl) propane]dihydrochloride.

Fig. 4. Functional characterization of 1

(A) CD spectrum of EPO(S)1 (1). (B) SDS-PAGE conducted on two separate gels (1, lanes a and b; 2, lanes c, d, and e) both using rEPOα (EMD) as control (lanes a and e). Lane b: EPO(S)1 (1); Lane c: EPO glycoform S3; Lane d: EPO (29-166) (9). (C) In vitro assay of the effect of Procrit and EPO(S)1 (1) on the proliferation of TF-1 cells. The results are expressed as average relative fluorescent intensity ± SD, run in triplicate. Relative fluorescent intensity = fluorescent intensity of EPO-treated TF-1 cells/maximal fluorescent intensity of 10 ng/ml Procrit-EPO–treated TF-1 cells. (D) Effect of Procrit and EPO(S)1 (1) on peripheral blood reticulocyte numbers in vivo. C57 mice (three mice per group) were subcutaneously injected with 100 μl of saline containing 1 mg/ml bovine serum albumin and various doses of EPO daily for 3 days. After 24 hours since the last injection, peripheral blood from each mouse was harvested and stained with reticulocyte stain reagent, and reticulocytes per 200 red blood cells were counted under a microscope with a 100× objective lens. The results are expressed as the average ± SD, n = 3 mice.