PMC

Retrosynthetic strategy toward EPO.

Earlier synthetic routes from our laboratory toward EPO.

Synthesis of glycopeptide 6 en route to EPO Fragment IV.

New Methods for the synthesis of proteins and glycoproteins. GP = glycopeptide.

Ribbon structure of erythropoietin containing a consensus sequence of N-linked carbohydrate domains.

The image of human BFU-E colony. 60 ng/ml Synthetic folded EPO (1). and 20 ng/ml rhKL stimulate purified cord blood CD34 cells to form BFU-E colony after 2 weeks.

Synthesis of EPO Fragment I (15). Reactions and conditions: (a) Chitobiose, HATU, DIPEA, DMSO; then TFA/TIS/H₂O/phenol, 45%. AA = side chain protected sequence. AA = pseudoproline dipeptide. Side chain protecting groups are listed in the .

Synthesis of EPO Fragment III (11). Reactions and conditions: (a) Chitobiose, HATU, DIPEA, DMSO; then TFA/TIS/H₂O/phenol, 40%. AA = side chain protected sequence. AA = pseudoproline dipeptide. Side chain protecting groups are listed in the .

Synthesis of EPO Fragment II (13). Reactions and conditions: (a) Chitobiose, HATU, DIPEA, DMSO; then TFA/TIS/H₂O/phenol, 54%.²⁷ AA = side chain protected sequence. AA = pseudoproline dipeptide. Side chain protecting groups are listed in the .

Synthesis of EPO Fragment IV (9). Reactions and Conditions: (a) 1. 6 M Gn·HCl, 100 mM Na₂HPO₄, 50 mM TCEP, pH 7.3, 2 h; 2. piperidine, DMSO, 10 min, 55% (two steps); (b) 0.2 M MeONH₂, 6 M Gn·HCl, 100 mM Na₂HPO₄, 50 mM TCEP, pH 4.0, 3 h, 75%.

(A) Top-down Mass Spectra of unfolded EPO. Calcd for C₈₉₉H₁₄₅₂N₂₄₂O₂₉₇S₅: 20562.0147 Da (average isotopes); observed: [M+12H]¹²⁺ m/z = 1715.8001, [M+14H]¹⁴⁺ m/z = 1582.7384, [M+15H]¹⁵⁺ m/z = 1371.7734, [M+16H]¹⁶⁺ m/z = 1286.0381, [M+17H]¹⁷⁺ m/z = 1210.6258, [M+18H]¹⁸⁺ m/z = 1143.4804, [M+19H]¹⁹⁺ m/z = 1083.2441, [M+20H]²⁰⁺ m/z = 1029.0825, [M+21H]²¹⁺ m/z = 980.1267, [M+22H]²²⁺ m/z = 935.7577, measured for C₈₉₉H₁₄₅₂N₂₄₂O₂₉₇S₅: (20562.1934± 0.1787) Da. (B) LC trace (UV) of folded erythropoietin chitobiose, and Top-down Mass Spectra of folded EPO. Calcd for C₈₉₉H₁₄₄₈N₂₄₂O₂₉₇S₅: 20557.9829 Da (average isotopes); observed: [M+11H]¹¹⁺ m/z = 1869.8790, [M+12H]¹²⁺ m/z = 1714.2245, [M+13H]¹³⁺ m/z = 1582.3602, [M+14H]¹⁴⁺ m/z = 1469.4066, [M+15H]¹⁵⁺ m/z = 1371.5131, [M+16H]¹⁶⁺ m/z = 1285.8580, [M+17H]¹⁷⁺ m/z = 1210.3956, [M+18H]¹⁸⁺ m/z = 1143.2618, measured for C₈₉₉H₁₄₄₈N₂₄₂O₂₉₇S₅: (20558.3348± 0.3518) Da.

Synthesis of non-glyco-EPO. Reaction conditions: a) Guanidine•HCl (6 M), Na₂HPO₄ (0.2 M), TCEP•HCl (0.02 M), MPAA (0.2 M), Bond-Breaker^®, pH 7.2–7.4; b) MeONH₂•HCl (0.3 M), pH 4.0, 34% yield over 2 steps; c) Guanidine•HCl (6 M), Na₂HPO₄ (0.2 M), TCEP•HCl (0.02 M), MPAA (0.2 M), Bond-Breaker^®, pH 7.2–7.4; 33% yield; d) Guanidine•HCl (6 M), Na₂HPO₄ (0.2 M), TCEP•HCl (0.08 M) pH 7.0, VA-044, tBuSH, 37 °C; e) AgOAc, CH₃CN/H₂O (1:1) + 0.05% TFA then DTT, 45% yield over 2 steps; f) Guanidine•HCl (6 M), Na₂HPO₄ (0.2 M), TCEP•HCl (0.02 M), MPAA (0.2 M), Bond-Breaker^®, pH 7.2–7.4, 35–40% yield; g) Tris•HCl (50 mM), CuSO₄ (40 mM), N-lauroylsarcosine (2% w/v), pH 8.0.

Synthesis of EPO primary structure (Ala¹-Arg¹⁶⁶). Reactions and conditions: (a) 6 M Gn•HCl, 300 mM Na₂HPO₄, 200 mM MPAA, 20 mM TCEP, pH 7.3, 18 h; then MeONH₂•HCl, DTT, 3 h, 72%; (b) 6 M Gn•HCl, 300 mM Na₂HPO₄, 200 mM MPAA, 20 mM TCEP, pH 7.3, 12 h, 74%; (c) 6 M Gn•HCl, 200 mM Na₂HPO₄, Bond-Breaker®, VA-044, tBuSH, 37 °C, 5 h, 68%; (d) AgOAc, HOAc/H₂O (1:1), 6 h, 62%; (e) 6 M Gn•HCl, 300 mM Na₂HPO₄, 200 mM MPAA, 20 mM TCEP, TFE, pH 7.3, 19 h, 73%..

Synthesis of folded EPO using convergent KCL. Reaction conditions: (a) 20 and 21, 6 M Gn•HCl, 150 mM Na₂HPO₄, 50 mM TCEP, pH 7.2, 5 h; then 16, 12 h; (b) 6 M Gn•HCl, 100 mM Na₂HPO₄, Bond-Breaker^®, VA-044, tBuSH, 37 °C, 10 h, 54% (three steps); (c) AgOAc, 70% AcOH, 6 h, 73%; (d) folding conditions: 50 mM Tris•HCl, 40 μM CuSO₄, N-lauroylsarcosine (2% w/v), pH 8.0.