Kilogram-Scale Synthesis of Osteogenic Growth Peptide (10–14) Using a Fragment Coupling Approach

Abstract

Kilogram-scale synthesis of a bioactive pentapeptide in solution by "3+2" fragment coupling strategy has been successively accomplished in the development of OGP (10-14), a minimal OGP-derived sequence that retains the full proliferative activity of the osteogenic growth peptide. The synthetic scheme, coupling conditions and scaling up of the process are systematically studied, the epimerization of the tripeptide fragment and pentapeptide are also evaluated. .65 (t, J = 7.6 Hz, 3H), 7.40 (t, J = 7.4 Hz, 3H), 7. 34 -7.27 (m, 3H), 7.25 (d, J = 4.1 Hz, 3H), 7.17 (d, J = 8.5 Hz, 2H), 4.56 (s, 1H), 4.19 (d, J = 11.2 Hz, 1H), 4.16 -4.06 (m, 3H), 3.89 -3.76 (m, 3H), 3.05 (dd, J = 13.9, 4.2 Hz, 1H), 2.95 (d, J = 9.8 Hz, 1H), 2.79 -2.68 (m, 2H), 1.41 -0.85 (m, 9H). Fmoc-Tyr(tBu)-Gly-Phe-OSu (3)To a stirred solution of Fmoc-Tyr(tBu)-Gly-Phe-OH (2.76 kg, 4.16mol) and HOSu (0.67 kg, 5.83mol, 1.4eq) in dry THF (25 L), a solution of DCC (1.21 kg, 5.83mol, 1.4eq) in dry THF (5 L) was added slowly under ice-cooling within 3h, then the solution was stirred overnight at ambient temperature.After cooling for 1 h, the resulting precipitate was filtered and filtrate was used further reaction without purification. TLC: a 2 , R f =0.3. Boc-Gly-OH (4)The mixture of glycine (1 kg, 13.32mol) and NaHCO 3 (1.12 kg, 13.32mmol) in H 2 O (15 L) was added slowly to the solution of Boc 2 O (3.06 L, 13.32mol) in THF (10 L) and stirred at ambient temperature for 5h. When TLC indicated the absence of starting material, the residue was evaporated and distributed by H 2 O (20 L), EtOAc (7 L) and light petroleum (7 L). The organic layer was removed and the aqueous phase adjusted to pH 2-3 with 10% citric acid solution. After extracted with EtOAc (3×10 L), the aqueous layer was separated and the combined organic extracts were washed with saline (2×10 L), dried with magnesium sulfate and evaporated to give a white crystal (1.98 kg, 84.85%). m.p.85.7～86.5°C, Boc-Gly-Gly-OH (5)Boc-Gly-OH (1.98 kg, 11.3mol) and HONp (1.73 kg, 12.43mol, 1.1eq) or HOSu (1.43 kg, 12.43mol, 1.1eq) were dissolved in dry 1,4-dioxane (25 L), a solution of DCC (2.8 kg, 13.56mol, 1.2eq) in dry 1,4-dioxane (10 L) was added and the mixture stirred for 8-10h. After cooling for 2h, the precipitate was removed, the filtrate was used directly for next reaction without purification, TLC: a 1 , R f =0.4, 0.3.The mixture of glycine (0.94 kg, 12.43mol, 1.1eq) and NaHCO 3 (1.05 kg, 12.43mol, 1.1eq) in H 2 O (10 L) was slowly added to the solution of Boc-Gly-ONp (get by above step) in 1,4-dioxane (25 L). The solution was stirred at about 25°C for 7 h, then evaporated under reduced pressure. The residue was acidified to a pH 2-3 with 10% citric acid and extracted with EtOAc (3×20 L). The organic phase was successively washed with saline (20 L), dried, and evaporated to give Boc-Gly-Gly-OH (2.37 kg, 90.3%) as a white schistose crystals. m.p.123.5～125.5°C, TLC: a 3 , R f =0.2, HPLC: e, 6.53min, purity 93.5%, Calc. for C 7 H 13 NO 4 : C, 46.55; H, 6.94; N, 12.06. Found: C, 45.40; H, 6.02; N, 14.13...

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Epimerization-Free C-Term Activation of Peptide Fragments by Ball Milling

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Scale-Up Synthesis and Identification of GLYX-13, a NMDAR Glycine-Site Partial Agonist for the Treatment of Major Depressive Disorder

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