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General procedure: Step 1: The general procedure described in Example 2 was followed for the coupling of Fmoc-AEEP-OH, Fmoc-Cys(ST3u)-OH, Fmoc- Asp(OiBu)-OH, and Fmoc-Dap(Ac)-OH to protected CCK8-Resin, 1. Resin cleavage and deprotection were performed under cleavage and purification conditions described in Example 2 to yield partially protected peptide, EC1943. Fmoc-Sieber-resin (l.Og, 0.69mmol) was placed in a peptide synthesis vessel, and washed with DMF (3 x 10 mL). Initial Fmoc deprotection was performed using 20% piperidine in DMF (3 x 10 mL) solution for 10 mins per cycle. The resin was further washed with DMF (3 x 10 mL) and z'-PrOH (3 x 10 mL), and a Kaiser test was conducted to determine that the reaction was complete. The resin was washed again with DMF wash (3 x 10 mL), and a solution of Fmoc-Phe-OH (0.57 g, 1.38 mmol, 2.0 eq.) in DMF, PyBOP (0.72 g, 1.38 mmol, 2.0 eq.) and DIPEA (0.37 mL, 2.07 mmol, 3.0 eq.) were added to the vessel. The resulting solution was bubbled with Argon for 1 hour. The coupling solution was filtered, the resin was washed with DMF (3 x 10 mL) and z'-PrOH (3 x 10 mL), and a Kaiser test was conducted to determine that the reaction was complete. The process was repeated for each additional coupling according to the reagent amounts listed in Table 1. Resin bound-protected CCK8 peptide, 1, (0.8g, 0.28mmol) was placed in a peptide synthesis vessel, and was subjected to solid phase synthesis as described in Example 1 for the coupling of Fmoc-AEEP-OH (<strong>[872679-70-4]Fmoc-9-amino-4,7-dioxanonanoic acid</strong>), Fmoc-Cys(Trt)-OH, Fmoc-Asp(OiBu)-OH, Boc-Dap(Fmoc)-OH (Na-Boc-^-Fmoc-L-2,3-diaminopropionic acid) and Ac20 using to the reagent amounts shown in Table 2. Resin cleavage was performed with a cocktail of 94% CF3C02H, 2.5% EDT, 2.0% triisopropylsilane and 1.5% H20. The cleavage cocktail (10 mL) was poured onto the resin and bubbled with Argon for 30 mins, followed by filtration into a clean flask. Further cleavage was performed two times with fresh cleavage cocktail and 10 mins of Argon bubbling. The combined filtrate was poured onto cold diethyl ether, and the precipitate that formed was collected by centrifugation at 4000 rpm for 5 mins (3x). The precipitate was obtained following decanting and drying of the solid under vacuum; the product was then purified by preparative HPLC (mobile phase A = lOmM Ammonium acetate, pH = 5; Organic phase B = Acetonitrile; Method; 10% B to 100%B in 30 mins) to yield EC1825 (2) (30 mg, 7%). 1H NMR (500 MHz DMSO- 6) Pivotal signals: delta 7.49 (d, J = 7.9 Hz, 1H), 7.31 (d, / = 8.1 Hz, 1H), 7.23 - 7.14 (m, 5H), 7.13 (s, 1H), 7.03 (t, J = 7.6 Hz, 1H), 6.96 - 6.89 (m, 2H), 6.60 (d, J = 8.4 Hz, 2H), 4.50 (dt, J = 9.1, 6.6 Hz, 2H), 4.42 - 4.33 (m, 2H), 4.28 (td, J = 9.8, 8.8, 4.7 Hz, 2H), 4.23 (dd, = 8.7, 5.2 Hz, 2H), 4.17 (dd, = 9.0, 5.0 Hz, 1H), 1.95 (s, 3H), 1.95 (s, 3H), 1.82 (s, 3H). [M+H]+ = Calculated 1567.6, found 1569.3 |
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