3.2. Procedures and Analytical Description of Compounds
Ester (11): To a suspension of carboxylic acid 10 (0.50 g, 1.2 mmol) in DCM (15 mL) was added DMF (0.93 μL, 0.012 mmol), followed by dropwise addition of oxalyl chloride (0.15 mL, 1.8 mmol) at 0 °C. The solution was allowed to warm to room temperature and stirred for 1.5 h. After the reaction was completed, the solvent was removed in vacuo to give the acid chloride that was used in the next step without further purification.
To a solution of the crude acid chloride in DCM (20 mL) was added 2-(trimethylsilyl) ethanol (0.25 mL, 1.8 mmol) carefully dropwise. The solution was stirred overnight at room temperature before it was quenched with 1 M HCl (5 mL). The organic layer was separated, washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography on silica gel, eluting with EtOAc/hexanes to afford the corresponding ester 11 in 84% yield (0.41 g, 1.0 mmol) as a white amorphous solid: Rf = 0.62 (ethyl acetate/hexanes = 1:2); = −0.90 (c 1.0, MeOH); 1H NMR (Methanol-d4, 500 MHz) δ 7.77 (2H, d, J = 7.5 Hz), 7.66 (2H, dd, J = 10.3, 7.5 Hz), 7.37 (2H, t, J = 7.4 Hz), 7.29 (2H, t, J = 7.5 Hz), 4.87 (1H, d, J = 2.6 Hz), 4.82 (1H, d, J = 2.5 Hz), 4.40 (1H, dd, J = 10.6, 6.9 Hz), 4.34 (1H, dd, J = 10.6, 7.0 Hz), 4.30–4.25 (1H, m), 4.25–4.17 (2H, m), 1.53 (3H, s), 1.51 (3H, s), 1.01 (2H, t, J = 8.6 Hz), 0.02 (9H, s); 13C NMR (Methanol-d4, 126 MHz) δ 171.5, 169.3, 158.5, 145.2, 145.1, 142.6, 142.5, 128.8, 128.2, 128.2, 126.3, 126.2, 120.9, 112.8, 76.8, 68.3, 65.5, 56.6, 48.3, 26.8, 26.1, 18.1, −1.6; HRMS (ESI) calculated for C27H33O7NSiNa+ [M + Na]+ 534.1920, found 534.1918.
Ester (4): The ester 11 (0.50 g, 1.2 mmol) was dissolved in a mixture of AcOH/H2O/THF 2:2:1 (10 mL) and heated at 45 °C for 12 h. The solvent was evaporated and the residue was azeotropically dried by co-evaporation from toluene (3 × 5 mL). The crude material was used immediately in the next step without further purification.
To a solution of the above carboxylic acid in anhydrous DMF (10 mL) at 0 °C under an Ar atmosphere was added DIPEA (0.26 mL, 1.5 mmol) and allyl bromide (0.13 mL, 1.5 mmol). The mixture was stirred at ambient temperature for 2 h and then quenched with H2O (5 mL). The product was extracted with EtOAc (2 × 30 mL) and the combined organic phases were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography on silica gel, eluting with EtOAc/hexanes to afford the corresponding allyl ester 4 in 70% yield (0.43 g, 0.84 mmol) as a white amorphous solid: Rf = 0.50 (ethyl acetate/hexanes = 1:2); = +1.80 (c 1.0, MeOH); 1H NMR (Methanol-d4, 500 MHz) δ 7.78 (2H, d, J = 7.3 Hz), 7.67 (2H, t, J = 6.9 Hz), 7.38 (2H, t, J = 7.4 Hz), 7.30 (2H, t, J = 7.3 Hz), 5.97 (1H, ddt, J = 16.7, 11.2, 5.8 Hz), 5.37 (1H, dt, J = 17.4, 1.6 Hz), 5.23 (1H, d, J = 10.5 Hz), 4.86 (1H, s), 4.81 (1H, d, J = 3.5 Hz), 4.68 (2H, d, J = 5.8 Hz), 4.50 (1H, d, J = 3.5 Hz), 4.40 (1H, dd, J = 10.4, 6.8 Hz), 4.35–4.28 (1H, m), 4.21 (3H, ddt, J = 13.1, 9.3, 4.8 Hz), 1.06–0.92 (2H, m), 0.03 (9H, s); 13C NMR (Methanol-d4, 126 MHz) δ 172.3, 170.4, 158.4, 145.2, 145.2, 142.6, 133.3, 128.8, 128.2, 126.3, 126.2, 120.9, 119.0, 72.9, 68.3, 67.1, 65.1, 58.7, 48.3, 18.2, −1.6; HRMS (ESI) calculated for C27H33O7NSiNa+ [M + Na]+ 534.1920, found 534.1918.
Dipeptide (13): To a stirred solution of allyl ester 4 (0.77 g, 1.07 mmol) in dichloromethane (20 mL) at 0 °C under an atmosphere of argon was added DBU (0.16 mL, 1.07 mmol). The reaction mixture was stirred for 1.5 h at 0 °C, at which point TLC analysis indicated that the starting material was consumed completely. Then, HOAt (0.22 g, 1.60 mmol) was added and the mixture was stirred for 5 min at 0 °C. Acid 5 (0.53 g, 1.18 mmol) and HATU (0.61 g, 1.60 mmol) were added, and the resulting homogeneous yellow solution was allowed to warm to room temperature and stirred overnight. The reaction mixture was cooled to 0 °C and quenched by the addition of H2O (10 mL) and extracted with EtOAc (2 × 30 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography on silica gel, eluting with EtOAc/hexanes to afford dipeptide 13 in 67% yield (0.52 g, 0.72 mmol) as a white amorphous solid: Rf = 0.54 (ethyl acetate/hexanes = 1:1); = −4.90 (c 1.0, MeOH); 1H NMR (Methanol-d4, 500 MHz) δ 7.79 (2H, dd, J = 7.7, 2.9 Hz), 7.70–7.63 (2H, m), 7.38 (2H, td, J = 7.5, 2.2 Hz), 7.31 (2H, t, J = 7.6 Hz), 5.97 (1H, ddt, J = 17.3, 11.8, 6.3 Hz), 5.37 (1H, d, J = 17.2 Hz), 5.24 (1H, d, J = 10.5 Hz), 5.03 (1H, d, J = 3.3 Hz), 4.81 (1H, s), 4.68 (2H, d, J = 5.8 Hz), 4.47 (1H, d, J = 3.3 Hz), 4.38 (2H, d, J = 7.1 Hz), 4.20 (4H, dt, J = 25.3, 6.2 Hz), 3.05 (2H, d, J = 7.1 Hz), 1.82 (1H, h, J = 5.5 Hz), 1.65 (1H, q, J = 5.1 Hz), 1.57–1.48 (1H, m), 1.43 (9H, s), 0.98 (2H, t, J = 8.7 Hz), 0.01 (9H, s); 13C NMR (Methanol-d4, 126 MHz) δ 174.6, 172.2, 169.8, 158.5, 145.3, 145.1, 142.6, 142.5, 133.2, 128.8, 128.2, 126.2, 126.2, 120.9, 119.0, 79.9, 72.5, 68.0, 67.1, 65.2, 56.8, 56.0, 40.8, 30.3, 28.8, 27.3, 18.2, −1.7; HRMS (ESI) calculated for C37H51O10N3SiNa+ [M + Na]+ 748.3237, found 748.3236.
Tripeptide (2): To a stirred solution of dipeptide 13 (0.38 g, 0.53 mmol) in dichloromethane (0 mL) at 0 °C under an atmosphere of argon was added DBU (78.5 μL, 0.53 mmol) via syringe. The reaction mixture was stirred for 1.5 h at 0 °C. After the starting material was consumed completely as determined by TLC, HOAt (0.11 g, 0.80 mmol) was added and the mixture was stirred for 5 min at 0 °C. Acid 6 (0.19 g, 0.53 mmol) followed by HATU (0.30 g, 0.80 mmol) were added and the resulting homogeneous yellow solution was allowed to warm to room temperature. The reaction mixture was cooled to 0 °C and quenched by the addition of H2O (5 mL) and extracted with EtOAc (2 × 15 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography on silica gel, eluting with EtOAc/hexanes to afford tripeptide 2 in 67% yield (0.30 g, 0.36 mmol) as a white amorphous solid: Rf = 0.55 (ethyl acetate/hexanes = 2:1); = −7.40 (c 1.0, MeOH); 1H NMR (Methanol-d4, 400 MHz) δ 7.80 (2H, d, J = 7.5 Hz), 7.68 (2H, t, J = 8.1 Hz), 7.39 (2H, t, J = 7.5 Hz), 7.31 (2H, td, J = 7.4, 1.2 Hz), 5.98 (1H, ddt, J = 16.4, 10.9, 5.7 Hz), 5.37 (1H, dt, J = 17.2, 1.6 Hz), 5.25 (1H, dq, J = 10.5, 1.4 Hz), 5.03 (1H, d, J = 3.3 Hz), 4.72–4.62 (2H, m), 4.51 (1H, dd, J = 8.9, 5.1 Hz), 4.47 (1H, d, J = 3.3 Hz), 4.44–4.34 (2H, m), 4.24 (1H, t, J = 6.9 Hz), 4.17 (3H, dt, J = 12.2, 4.5 Hz), 3.03 (2H, q, J = 6.6 Hz), 1.90 (2H, tt, J = 13.7, 6.9 Hz), 1.66 (1H, td, J = 8.9, 4.6 Hz), 1.58–1.45 (3H, m), 1.40 (9H, s), 1.25–1.17 (1H, m), 1.01–0.83 (8H, m), 0.02 (9H, s); 13C NMR (Methanol-d4, 101 MHz) δ 174.6, 173.8, 172.2, 169.7, 158.8, 158.5, 145.4, 145.1, 142.5, 133.2, 128.7, 128.1, 128.1, 126.2, 120.9, 118.9, 79.9, 72.5, 68.0, 67.1, 65.1, 60.4, 56.8, 54.0, 48.4, 40.7, 38.2, 30.2, 28.7, 27.2, 18.2, 15.0, 11.9, −1.7; HRMS (ESI) calculated for C43H62O11N4SiNa+ [M + Na]+ 861.4077, found 861.4077.
Dipeptide (16): To a stirred solution of N-Boc-d-valine (1.24 g, 5.71 mmol) and d-phenylalanine allyl ester 7 (1.23 g, 6.0 mmol) in DMF (50 mL) at 0 °C under an atmosphere of argon was added DIPEA (2.98 mL, 17.13 mmol) and HOAt (1.17 g, 8.57 mmol), and the mixture was stirred for 5 min. HATU (3.25 g, 8.57 mmol) was then added, and the resulting homogeneous reaction mixture was allowed to warm to room temperature. The reaction mixture was cooled to 0 °C, quenched by the addition of H2O (10 mL) and extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with aqueous 1 N HCl (2 × 30 mL), saturated aqueous NaHCO3 (2 × 30 mL), H2O (30 mL), and brine (30 mL) and dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography on silica gel, eluting with EtOAc/hexanes to afford dipeptide 16 in 84% yield (1.93 g, 4.80 mmol) as a white amorphous solid: Rf = 0.55 (ethyl acetate/hexanes = 1:2); = +19.30 (c 1.0, MeOH); 1H NMR (Methanol-d4, 500 MHz) δ 7.29–7.16 (5H, m), 5.85 (1H, ddt, J = 16.4, 10.9, 5.7 Hz), 5.30–5.24 (1H, m), 5.19 (1H, dd, J = 10.6, 1.5 Hz), 4.71 (1H, dd, J = 8.5, 6.0 Hz), 4.56 (2H, dq, J = 5.5, 1.8 Hz), 3.86 (1H, d, J = 7.2 Hz), 3.15 (1H, dd, J = 13.9, 6.1 Hz), 3.02 (1H, dd, J = 13.8, 8.4 Hz), 1.94 (1H, dt, J = 13.9, 7.0 Hz), 1.43 (9H, s), 0.87 (6H, dd, J = 10.2, 6.8 Hz); 13C NMR (Methanol-d4, 126 MHz) δ 174.3, 172.4, 157.8, 138.0, 133.2, 130.3, 129.5, 127.9, 118.9, 80.5, 66.8, 61.4, 55.1, 38.5, 32.1, 28.7, 19.7, 18.5; HRMS (ESI) calculated for C22H32O5N2Na+ [M + Na]+ 427.2205, found 427.2203.
l-5-chloro-tryptophan hydrochloride (15): In a 100 mL round-bottomed flask, 5-Cl-indole (773.11 mg, 5.10 mmol), l-serine 14 (589.55 mg, 5.61 mmol) and pyridoxal phosphate (26.52 mg, 0.10 mmol) were suspended in DMSO (1.0 mL) and 50 mM KPi buffer (20 mL). The vial was sealed and then placed in a water bath that had been equilibrated to 70 °C. After 1 min, the enzyme was added. The reaction was kept at 70 °C. After 12 h, the reaction mixture was cooled in an ice bath for 90 min. The precipitate was collected by filtration and washed with ethyl acetate (2 × 20 mL) and ice water (2 × 20 mL). The precipitate was dissolved in a mixture of 1 M hydrochloric acid and acetonitrile (1:1), filtered, and the filtrate was concentrated in vacuo to afford l-5-chloro-tryptophan hydrochloride 15 in 50% yield (698.78 mg, 2.55 mmol) as a white amorphous solid: = +6.70 (c 1.0, MeOH); 1H NMR (400 MHz, DMSO-d6) δ 11.38 (d, J = 2.7 Hz, 1H), 8.43 (s, 3H), 7.64 (d, J = 2.0 Hz, 1H), 7.39 (d, J = 8.6 Hz, 1H), 7.34 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 8.6, 2.1 Hz, 1H), 4.10 (t, J = 6.0 Hz, 1H), 3.28 (d, J = 5.9 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 170.8, 134.8, 128.4, 127.0, 123.5, 121.2, 117.8, 113.2, 106.8, 52.7, 25.8; HRMS (ESI) calculated for C11H11ClN2O2H+ [M + H]+ 239.0585, found 239.0582.
Teoc-protected amino acid (9): A mixture of Teoc-OSu (1.04 g, 4.0 mmol), 15 (0.95 g, 4.0 mmol), and Et3N (0.17 mL, 1.2 mmol) in 1:1 H2O-dioxane (40 mL) was stirred for 16 h before it was diluted with 1 N KHSO4 (10 mL). The aqueous layer was extracted with ether (4 × 50 mL). The combined organic layers were washed with water (2 × 20 mL), dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography on silica gel, eluting with EtOAc/hexanes, providing the Teoc-protected amino acid 9 in 96% yield (1.47 g, 3.84 mmol) as a white amorphous solid: Rf = 0.55 (ethyl acetate/hexanes = 1:1); = −3.50 (c 1.0, MeOH); 1H NMR (Methanol-d4, 500 MHz) δ 7.54 (1H, d, J = 2.0 Hz), 7.27 (1H, d, J = 8.6 Hz), 7.13 (1H, s), 7.04 (1H, dd, J = 8.5, 2.0 Hz), 4.47 (1H, dd, J = 7.8, 5.2 Hz), 4.15–4.00 (2H, m), 3.29 (1H, d, J = 5.0 Hz), 3.12 (1H, dd, J = 14.7, 7.8 Hz), 0.93 (2H, t, J = 8.5 Hz), −0.00 (9H, s); 13C NMR (Methanol-d4, 126 MHz) δ 175.5, 158.6, 136.3, 130.1, 126.6, 126.2, 125.6, 122.5, 118.8, 113.5, 111.0, 64.2, 56.2, 28.5, 18.6, −1.5; HRMS (ESI) calculated for C17H23O4N2ClSiNa+ [M + Na]+ 405.1008, found 405.1008.
Tripeptide (3): To a solution of compound 16 (40.42 mg, 0.1 mmol) in dry DCM (2 mL) at 0 °C was added Trifluoroacetic acid (1 mL). The reaction mixture was stirred at room temperature for 2 h. Volatiles were removed in vacuo and the residue was dried under high vacuum conditions for 2 h to afford the corresponding amine, which was used in the next step without further purification.
To a solution of amino acid 9 (41.41 mg, 0.1 mmol) in DCM (5 mL) at 0 °C were added DIPEA (69.68 μL, 0.4 mmol) and HOAt (20.42 mg, 0.15 mmol), and the mixture was stirred for 5 min. HATU (57.03 mg, 0.15 mmol) was added and the resulting homogeneous reaction mixture was allowed to warm to room temperature. The reaction mixture was cooled to 0 °C and quenched by the addition of H2O (2 mL) and extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with aqueous 1 N HCl (2 × 10 mL), saturated aqueous NaHCO3 (2 × 10 mL), H2O (10 mL), and brine (10 mL), dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography on silica gel, eluting with EtOAc/hexanes to afford tripeptide 3 in 74% yield over two steps (0.049 g, 0.074 mmol) as a white amorphous solid: Rf = 0.56 (ethyl acetate/hexanes = 2:1); = +13.20 (c 1.0, MeOH); 1H NMR (Methanol-d4, 500 MHz) δ 7.61–7.57 (1H, m), 7.30–7.17 (5H, m), 7.20–7.13 (1H, m), 7.12 (1H, s), 7.04 (1H, dd, J = 8.7, 2.1 Hz), 5.85 (1H, ddt, J = 16.4, 10.9, 5.7 Hz), 5.26 (1H, dd, J = 17.2, 1.7 Hz), 5.18 (1H, dd, J = 10.5, 1.5 Hz), 4.66 (1H, dt, J = 9.4, 5.6 Hz), 4.55 (2H, dd, J = 5.6, 1.4 Hz), 4.42 (1H, t, J = 7.3 Hz), 4.20–4.00 (3H, m), 3.21–3.13 (2H, m), 3.03 (2H, dt, J = 14.2, 8.9 Hz), 1.99–1.86 (1H, m, J = 6.8 Hz), 0.96 (2H, t, J = 8.6 Hz), 0.51 (6H, dd, J = 11.9, 6.8 Hz), 0.00 (9H, s); 13C NMR (Methanol-d4, 126 MHz) δ 174.9, 173.5, 172.4, 158.7, 138.2, 136.4, 133.2, 130.3, 129.9, 129.5, 127.8, 126.5, 125.6, 122.6, 119.0, 118.8, 113.5, 110.7, 66.8, 64.3, 59.8, 57.5, 55.3, 38.2, 31.0, 28.6, 19.3, 18.6, 17.7, −1.5; HRMS (ESI) calculated for C34H45O6N4ClSiNa+ [M + Na]+ 691.2691, found 691.2689.
Hexapeptide (17): To a solution of allyl ester 3 (80.19 mg, 0.12 mmol) in dry THF (5 mL) was added Pd(PPh3)4 (6.93 mg, 0.006 mmol), followed by the dropwise addition of morpholine (0.1 mL, 1.20 mmol). The reaction mixture was stirred for 45 min at ambient temperature, and then concentrated. The residue was dissolved in DCM (30 mL) and washed with 1 N HCl (10 mL) and H2O (10 mL). The organic layer was dried with anhydrous Na2SO4 and concentrated. The crude acid intermediate S1 was obtained as an oil which was used in the next step without further purification. To a solution of Tripeptide 2 (100.61 mg, 0.12 mmol) in dichloromethane (10 mL) at 0 °C was added DBU (80.88 μL, 0.12 mmol), and stirred for 1.5 h. After the starting material was consumed completely as determined by TLC, HOAt (24.50 mg, 0.18 mmol) was added and the mixture was stirred for 5 min. Then, S1 (78.97 mg, 0.12 mmol) and HATU (68.44 mg, 0.18 mmol) were added, and the resulting homogeneous yellow solution was allowed to warm to room temperature. The reaction mixture was cooled to 0 °C, quenched by the addition of H2O (2 mL), and extracted with EtOAc (2 × 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography on silica gel, eluting with EtOAc/hexanes to afford hexapeptide 17 in 55% yield over two steps (0.081 g, 0.066 mmol) as a white amorphous solid: Rf = 0.31 (methanol/dichloromethane = 1: 20); = −13.80 (c 1.0, MeOH:DCM = 1:1); 1H NMR (DMSO-d6, 500 MHz) δ 11.00 (1H, d, J = 2.4 Hz), 8.17 (2H, t, J = 10.0 Hz), 7.99 (2H, dd, J = 12.7, 8.4 Hz), 7.83 (1H, d, J = 8.9 Hz), 7.70 (1H, s), 7.32 (1H, d, J = 8.7 Hz), 7.25–7.19 (5H, m), 7.14 (2H, dd, J = 12.9, 7.7 Hz), 7.03 (1H, dd, J = 8.6, 2.0 Hz), 6.74 (1H, t, J = 5.8 Hz), 6.05 (1H, s), 5.93 (1H, ddt, J = 16.2, 10.7, 5.6 Hz), 5.35 (1H, dt, J = 17.3, 1.7 Hz), 5.23 (1H, dd, J = 10.4, 1.6 Hz), 4.85 (1H, dd, J = 8.6, 3.8 Hz), 4.71–4.62 (1H, m), 4.61 (2H, dd, J = 5.4, 2.8 Hz), 4.40 (1H, td, J = 8.5, 5.4 Hz), 4.34 (2H, td, J = 8.8, 5.1 Hz), 4.27 (1H, d, J = 3.8 Hz), 4.16 (1H, t, J = 7.7 Hz), 4.13–4.01 (2H, m), 3.94 (2H, t, J = 8.5 Hz), 3.02 (1H, dd, J = 14.4, 5.1 Hz), 2.96 (1H, dd, J = 13.7, 5.8 Hz), 2.93–2.78 (4H, m), 2.00–1.90 (1H, m), 1.76 (1H, p, J = 6.6 Hz), 1.68–1.59 (1H, m), 1.50 (1H, d, J = 10.5 Hz), 1.36 (11H, s), 1.15 (1H, dd, J = 13.9, 6.8 Hz), 0.94–0.88 (3H, m), 0.84 (2H, td, J = 7.9, 2.8 Hz), 0.77 (3H, t, J = 7.3 Hz), 0.68 (11H, dt, J = 10.3, 3.9 Hz), −0.02 (18H, d, J = 26.5 Hz); 13C NMR (DMSO-d6, 126 MHz) δ 172.0, 171.6, 170.9, 170.8, 170.6, 170.4, 168.5, 156.1, 155.6, 137.5, 134.5, 132.3, 129.1, 128.5, 128.0, 126.1, 125.9, 123.0, 120.7, 118.0, 117.9, 112.7, 110.1, 77.4, 71.3, 65.0, 63.0, 61.8, 57.4, 55.5, 54.9, 54.2, 52.0, 37.9, 37.0, 31.3, 30.3, 29.3, 29.0, 28.3, 27.8, 25.9, 25.6, 19.1, 17.6, 17.3, 16.8, 14.2, 11.6, −1.6, −1.6; HRMS (ESI) calculated for C59H91N8O14Si2ClNa+ [M + Na]+ 1249.5779, found 1249.5774.
Hexapeptide (18): To a solution of the hexapeptide 17 (355.71 mg, 0.29 mmol) in THF (5 mL) was added 1.0 M TBAF in THF (2.9 mL, 2.9 mmol). The reaction mixture was stirred at 50 °C for 1 h and then cooled to 0 °C and quenched by the addition of a saturated aqueous solution of NH4Cl (5 mL). The reaction mixture was transferred to a separatory funnel and extracted with EtOAc (3 × 30 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The crude product was used in the next step without further purification.
To a solution of the above crude product in a mixture of dichloromethane (280 mL) and DMF (15 mL) were sequentially added DMAP (7.09 mg, 0.058 mmol), HOAt (78.94 mg, 0.58 mmol), and EDCI (111.19 mg, 0.58 mmol). The reaction mixture was stirred at 30 °C for 48 h and then cooled to 0 °C and quenched by the addition of a saturated aqueous solution of NH4Cl (20 mL). The mixture was transferred to a separatory funnel where the aqueous layer was extracted with DCM (3 × 30 mL). The combined organic layers were washed with H2O (2 × 50 mL) and brine (50 mL), dried over Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel, eluting with MeOH/DCM to afford hexapeptide 18 in 65% yield (0.18 g, 0.19 mmol) as a white amorphous solid: Rf = 0.33 (methanol/dichloromethane = 1:20); = +6.60 (c 1.0, MeOH:DCM = 1:1); 1H NMR (DMSO-d6, 400 MHz) δ 11.02 (1H, d, J = 2.5 Hz), 8.17 (1H, d, J = 7.2 Hz), 7.87 (2H, dd, J = 8.0, 4.2 Hz), 7.81 (1H, d, J = 7.2 Hz), 7.73 (1H, dd, J = 15.7, 8.6 Hz), 7.54 (1H, d, J = 2.0 Hz), 7.46 (1H, d, J = 6.7 Hz), 7.34–7.08 (7H, m), 7.03 (1H, dd, J = 8.6, 2.1 Hz), 6.77 (1H, t, J = 5.8 Hz), 6.05 (1H, dd, J = 6.4, 2.0 Hz), 6.01–5.83 (1H, m), 5.32 (1H, dq, J = 17.3, 1.7 Hz), 5.20 (1H, dq, J = 10.5, 1.5 Hz), 4.66 (2H, q, J = 7.0, 6.5 Hz), 4.64–4.42 (3H, m), 4.27 (1H, td, J = 7.9, 7.3, 3.0 Hz), 4.17–4.09 (1H, m), 3.96–3.88 (1H, m), 3.88–3.78 (1H, m), 3.11 (1H, dd, J = 14.2, 7.9 Hz), 3.04–2.80 (5H, m), 1.90–1.66 (2H, m), 1.50 (1H, d, J = 8.2 Hz), 1.35 (10H, s), 1.24 (1H, d, J = 10.1 Hz), 1.01–0.78 (3H, m), 0.75–0.64 (6H, m), 0.55 (6H, dt, J = 17.9, 6.5 Hz); 13C NMR (DMSO-d6, 101 MHz) δ 171.9, 171.4, 171.1, 171.0, 170.9, 170.7, 168.6, 155.5, 137.1, 137.0, 134.5, 132.4, 129.1, 128.3, 128.1, 126.3, 125.7, 123.0, 120.7, 117.9, 117.5, 113.3, 112.8, 109.5, 77.3, 70.6, 65.0, 61.8, 59.5, 57.6, 54.9, 54.5, 53.7, 52.6, 37.9, 35.8, 29.5, 28.2, 28.1, 26.7, 26.3, 25.4, 18.9, 17.9, 14.2, 11.5; HRMS (ESI) calculated for C48H65O11N8ClNa+ [M + Na]+ 987.4351, found 987.4354.
Noursamycin A (1): Cyclic hexapeptide 18 (28.93 mg, 0.03 mmol) was dissolved in a saturated methanolic NH3 solution (10 mL) and stirred at 0 °C. After the starting material was consumed completely as determined by TLC, the reaction mixture was concentrated. The residue was azeotropically dried by concentration from methanol (2 × 10 mL) to remove ammonia from the product. The residue was used in the next step without further purification.
Trifluoroacetic acid (1 mL) was added to a solution of the above amide (27.70 mg, 0.03 mmol) in dry DCM (3 mL) at 0 °C. The reaction mixture was stirred for 0.5 h at rt. Volatiles were removed in vacuo and the residue was purified by column chromatography on silica gel, eluting with MeOH/DCM to afford 1 in 58% yield over two steps (14.33 mg, 0.017 mmol) as a white amorphous solid: Rf = 0.23 (methanol/dichloromethane = 1:2); = +11.40 (c 1.0, MeOH); IR (MeOH) 3472, 3385, 2967, 2073, 1632, 1545, 1205, 1053, 1016 cm−1; 1H NMR (DMSO-d6, 500 MHz) δ 11.02 (1H, s), 8.09 (1H, d, J = 8.2 Hz), 7.97 (2H, d, J = 8.0 Hz), 7.81 (1H, d, J = 7.5 Hz), 7.76 (2H, d, J = 6.0 Hz), 7.76–7.69 (2H, m), 7.59 (1H, s), 7.48 (1H, s), 7.33 (2H, dd, J = 19.4, 8.1 Hz), 7.25 (2H, t, J = 7.5 Hz), 7.16 (3H, dd, J = 12.1, 7.2 Hz), 7.12 (1H, s), 7.08–7.00 (1H, m), 6.99 (1H, s), 4.79 (1H, q, J = 7.3 Hz), 4.56 (1H, q, J = 7.9 Hz), 4.50 (2H, s), 4.18 (1H, q, J = 8.0, 7.6 Hz), 3.89–3.79 (2H, m), 3.31 (1H, dd, J = 14.9, 5.3 Hz), 3.01–2.90 (2H, m), 2.73 (3H, dd, J = 15.2, 9.0 Hz), 2.01–1.92 (1H, m), 1.86 (1H, hept, J = 6.9 Hz), 1.72 (1H, h, J = 6.6 Hz), 1.55 (1H, dd, J = 15.5, 9.0 Hz), 1.51–1.42 (2H, m), 0.91 (2H, dtt, J = 29.3, 15.5, 7.4 Hz), 0.74–0.68 (9H, m), 0.65 (3H, d, J = 6.6 Hz); 13C NMR (DMSO-d6, 126 MHz) δ 174.2, 172.1, 171.4, 171.1, 170.7, 170.6, 169.0, 137.2, 134.5, 129.3, 128.3, 128.1, 126.3, 125.6, 123.0, 120.7, 117.3, 112.8, 110.1, 70.6, 60.6, 58.2, 56.6, 53.9, 52.6, 52.4, 38.5, 35.8, 29.7, 27.7, 26.1, 25.4, 24.2, 19.1, 18.8, 14.3, 11.6; HRMS (ESI) calculated for C40H55N9O8Cl+ [M + H]+ 824.3857, found 824.3857.
Ester (S2): To a suspension of carboxylic acid 19 (2.76 g, 6.7 mmol) in DCM (40 mL), oxalyl chloride (0.68 mL, 8.0 mmol) was added dropwise at 0 °C, followed by the addition of DMF (5.67 μL, 0.067 mmol). The solution was allowed to warm to room temperature and stirred continuously for 2 h. After the reaction was completed, the solvent was removed in vacuo to give the acid chloride that was used in the next step without additional purification.
To a solution of the crude acid chloride in DCM (20 mL) was added 2-(trimethylsilyl) ethanol (1.05 mL, 7.4 mmol) dropwise. The mixture was stirred overnight at room temperature before it was quenched with 1 M HCl (5 mL). The organic layer was separated, washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography on silica gel, eluting with EtOAc/hexanes to afford the corresponding ester S2 in 79% yield (2.71 g, 5.3 mmol) as a white amorphous solid: Rf = 0.66 (ethyl acetate/hexanes = 1:2); = +11.10 (c 1.0, MeOH); 1H NMR (Methanol-d4, 500 MHz) δ 7.79 (2H, d, J = 7.5 Hz), 7.66 (2H, d, J = 7.5 Hz), 7.39 (2H, t, J = 7.3 Hz), 7.30 (2H, tt, J = 7.5, 1.5 Hz), 5.10 (1H, d, J = 2.4 Hz), 4.40–4.31 (2H, m), 4.33–4.26 (2H, m), 4.24 (1H, t, J = 7.1 Hz), 1.66 (3H, s), 1.57 (3H, s), 1.29 (1H, d, J = 6.9 Hz), 1.09–1.01 (2H, m), 0.05 (9H, s); 13C NMR (Methanol-d4, 126 MHz) δ 171.7, 170.3, 158.6, 145.2, 145.1, 142.6, 128.8, 128.2, 126.3, 126.3, 120.9, 112.9, 76.4, 68.5, 65.6, 55.6, 48.3, 26.7, 26.0, 18.2, −1.5; HRMS (ESI) calculated for C27H33O7NSiNa+ [M + Na]+ 534.1920, found 534.1918.
Ester (20): The ester S2 (2.45 g, 4.8 mmol) was dissolved in a mixture of AcOH/H2O/THF 2:2:1 (50 mL) and heated to 45 °C for 12 h. The solvent was evaporated, and the residue was azeotropically dried by concentration from toluene (3 × 5 mL). The crude material was submitted to the following step without purification.
The above carboxylic acid was dissolved in anhydrous DMF (30 mL) and cooled to 0 °C under an Ar atmosphere. Then, DIPEA (1.35 mL, 7.2 mmol) and allyl bromide (0.62 mL, 7.2 mmol) were added sequentially. The mixture was left to stir at ambient temperature for 2 h. The reaction was quenched with H2O (10 mL) and the product was extracted with EtOAc (2 × 50 mL). The combined organic phases were washed with brine (20 mL), dried (Na2SO4), filtered, and concentrated. The crude product was purified by column chromatography on silica gel, eluting with EtOAc/hexanes to afford the corresponding allyl ester 20 in 72% yield over two steps (1.77 g, 3.5 mmol) as a white amorphous solid: Rf = 0.43 (ethyl acetate/hexanes = 1:2); = +4.40 (c 1.0, MeOH); 1H NMR (Methanol-d4, 500 MHz) δ 7.78 (2H, d, J = 7.5 Hz), 7.64 (2H, t, J = 7.1 Hz), 7.38 (2H, tt, J = 7.7, 1.5 Hz), 7.30 (2H, tdd, J = 7.5, 4.4, 1.2 Hz), 5.89 (1H, ddt, J = 16.5, 11.1, 5.7 Hz), 5.29 (1H, dq, J = 17.3, 1.5 Hz), 5.15 (1H, dd, J = 10.6, 1.5 Hz), 4.80–4.74 (2H, m), 4.63 (1H, d, J = 4.0 Hz), 4.64–4.57 (1H, m), 4.32 (2H, d, J = 7.1 Hz), 4.32–4.22 (2H, m), 4.21 (1H, t, J = 7.1 Hz), 1.07–0.98 (2H, m), 0.05 (9H, s); 13C NMR (Methanol-d4, 126 MHz) δ 172.4, 171.0, 158.5, 145.2, 145.1, 142.6, 133.1, 128.8, 128.2, 128.2, 126.3, 120.9, 119.0, 72.2, 68.4, 67.2, 65.3, 58.6, 48.2, 18.2, −1.5; HRMS (ESI) calculated for C27H33O7NSiNa+ [M + Na]+ 534.1920, found 534.1918.
Dipeptide (S4): Allyl ester 20 (0.61 g, 1.2 mmol) was added to a 50 mL round-bottom flask equipped with a Teflon stir bar and fitted with a septum under an atmosphere of Ar. DCM (20 mL) was added via syringe and the mixture was stirred until it became homogeneous and colorless. The solution was cooled to 0 °C using an ice bath, and DBU (0.17 mL, 1.2 mmol) was added via syringe. The reaction mixture was stirred for 1.5 h at 0 °C. After the starting material was consumed completely as determined by TLC, HOAt (0.25 g, 1.8 mmol) was added and the mixture was stirred for 5 min at 0 °C using an ice bath. Acid 5 (0.55 g, 1.2 mmol) followed by HATU (0.68 g, 1.8 mmol) were added and the resulting homogeneous yellow reaction mixture was warmed to room temperature and stirred overnight. After 12 h, the reaction mixture was cooled to 0 °C using an ice bath and quenched by the addition of H2O (20 mL). The mixture was transferred with H2O (20 mL) to a separatory funnel and extracted with EtOAc (2 × 50 mL). The combined organic layers were dried over Na2SO4. Following the removal of the solvent that provided a white solid, the crude product was purified by column chromatography on silica gel, eluting with EtOAc/hexanes to afford dipeptide S4 in 60% yield (0.52 g, 0.72 mmol) as a white amorphous solid: Rf = 0.54 (ethyl acetate/hexanes = 1:1); = −4.70 (c 1.0, MeOH); 1H NMR (Methanol-d4, 500 MHz) δ 7.79 (2H, d, J = 7.5 Hz), 7.67 (2H, dd, J = 15.1, 7.5 Hz), 7.38 (2H, t, J = 7.4 Hz), 7.30 (2H, t, J = 7.5 Hz), 5.93 (1H, ddt, J = 16.5, 11.0, 5.8 Hz), 5.37–5.28 (1H, m), 5.22 (1H, d, J = 10.5 Hz), 4.96 (1H, d, J = 2.5 Hz), 4.76 (1H, d, J = 2.7 Hz), 4.63 (2H, d, J = 5.9 Hz), 4.36 (2H, qd, J = 10.7, 7.8 Hz), 4.24 (3H, ddt, J = 10.9, 7.7, 3.9 Hz), 4.17 (1H, dd, J = 8.9, 5.3 Hz), 3.05 (2H, t, J = 6.8 Hz), 1.76 (1H, td, J = 11.9, 9.8, 5.5 Hz), 1.61 (1H, qd, J = 9.5, 6.3 Hz), 1.57–1.48 (2H, m), 1.43 (10H, s), 1.02 (2H, dd, J = 9.7, 7.5 Hz), 0.02 (9H, s); 13C NMR (Methanol-d4, 126 MHz) δ 174.9, 172.4, 170.4, 158.5, 158.4, 145.5, 145.1, 142.6, 142.5, 133.2, 128.8, 128.7, 128.2, 126.3, 126.2, 120.9, 119.1, 80.0, 71.9, 68.1, 67.3, 65.3, 56.5, 56.2, 48.4, 40.8, 30.5, 28.8, 27.2, 18.2, −1.6; HRMS (ESI) calculated for C37H51O10N3SiNa+ [M + Na]+ 748.3238, found 748.3236.
Tripeptide (21): Under an atmosphere of Ar, dipeptide S4 (0.78 g, 1.07 mmol) was added to a 50 mL round-bottom flask equipped with a Teflon stir bar and fitted with a septum. DCM (20 mL) was added via syringe and the mixture was stirred until it became homogeneous and colorless. The solution was cooled to 0 °C using an ice bath, and DBU (0.16 mL, 1.07 mmol) was added via syringe. The reaction mixture was stirred for 1.5 h at 0 °C. After the starting material was consumed completely as determined by TLC, HOAt (0.22 g, 1.60 mmol) was added and the mixture was stirred for 5 min at 0 °C using an ice bath. Acid 6 (0.38 g, 1.07 mmol) followed by HATU (0.61 g, 1.60 mmol) were added and the resulting homogeneous yellow reaction mixture was warmed to room temperature. After 12 h, the reaction mixture was cooled to 0 °C using an ice bath and quenched by the addition of H2O (5 mL). The mixture was transferred with H2O (10 mL) to a separatory funnel and extracted with EtOAc (2 × 40 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography on silica gel, eluting with EtOAc/hexanes to afford tripeptide 21 in 65% yield (0.59 g, 0.70 mmol) as a white amorphous solid: Rf = 0.57 (ethyl acetate/hexanes = 1:1); = −7.10 (c 1.0, MeOH); 1H NMR (Methanol-d4, 500 MHz) δ 7.79 (2H, d, J = 7.5 Hz), 7.68 (2H, dd, J = 12.1, 7.5 Hz), 7.39 (2H, t, J = 7.5 Hz), 7.31 (2H, t, J = 7.4 Hz), 5.94 (1H, ddt, J = 16.5, 11.0, 5.8 Hz), 5.34 (1H, dq, J = 17.0, 1.5 Hz), 5.24 (1H, dd, J = 10.4, 1.5 Hz), 4.97 (1H, d, J = 2.8 Hz), 4.76 (1H, d, J = 2.8 Hz), 4.62 (2H, t, J = 5.4 Hz), 4.56 (1H, s), 4.49 (1H, dd, J = 8.7, 5.1 Hz), 4.39 (2H, qd, J = 10.6, 6.9 Hz), 4.27–4.18 (3H, m), 4.14 (1H, d, J = 5.7 Hz), 3.02 (2H, td, J = 6.9, 4.8 Hz), 1.92 (1H, p, J = 6.7 Hz), 1.81 (1H, dt, J = 14.5, 7.4 Hz), 1.62 (1H, dtd, J = 14.3, 9.2, 5.5 Hz), 1.54–1.45 (2H, m), 1.40 (9H, s), 1.20 (1H, tt, J = 14.8, 6.5 Hz), 1.01 (2H, t), 0.97–0.84 (6H, m), 0.06 (9H, s); 13C NMR (Methanol-d4, 126 MHz) δ 174.4, 174.0, 172.4, 170.4, 158.9, 158.5, 145.4, 145.1, 142.6, 133.2, 128.8, 128.2, 128.2, 126.3, 120.9, 119.2, 79.9, 72.0, 68.1, 67.3, 65.3, 60.4, 56.5, 54.0, 48.4, 40.8, 38.2, 30.5, 28.8, 27.3, 27.2, 18.2, 15.0, 12.0, −1.6; HRMS (ESI) calculated for C43H62O11N4SiNa+ [M + Na]+ 861.4077, found 861.4076.
Dipeptide (S5): Under an atmosphere of Ar, N-Boc-d-valine (0.36 g, 1.66 mmol) and d-leucine allyl ester 22 (0.28 g, 1.66 mmol) were added to a 100 mL round-bottom flask equipped with a Teflon stir bar and fitted with a septum. DMF (30 mL) was added via syringe and the mixture was stirred until it became homogeneous and colorless. The solution was cooled to 0 °C using an ice bath, and DIPEA (1.16 mL, 6.64 mmol) and HOAt (0.34 g, 2.49 mmol) were added and the mixture was stirred for 5 min. HATU (0.95 g, 2.49 mmol) was then added, and the resulting homogeneous reaction solution was warmed to room temperature. After 12 h, the reaction mixture was cooled to 0 °C using an ice bath and quenched by the addition of H2O (10 mL). The mixture was transferred with H2O (20 mL) to a separatory funnel and extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with aqueous 1 N HCl (2 × 30 mL), brine (2 × 30 mL), H2O (30 mL), and brine (30 mL), dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography on silica gel, eluting with EtOAc/hexanes to afford dipeptide S5 in 80% yield (0.49 g, 1.33 mmol) as a white amorphous solid: Rf = 0.74 (ethyl acetate/hexanes = 1:2); = +29.60 (c 1.0, MeOH); 1H NMR (Chloroform-d, 500 MHz) δ 6.47 (1H, d, J = 7.7 Hz), 5.86 (1H, ddt, J = 16.4, 10.9, 5.7 Hz), 5.29 (1H, d, J = 17.1 Hz), 5.21 (1H, d, J = 10.4 Hz), 5.14 (1H, d, J = 9.0 Hz), 4.59 (3H, t, J = 7.0 Hz), 3.90 (1H, t, J = 7.8 Hz), 2.11–2.02 (1H, m), 1.63 (2H, ddd, J = 13.4, 8.5, 5.3 Hz), 1.54 (1H, q, J = 8.9 Hz), 1.40 (9H, s), 0.93 (3H, d, J = 6.8 Hz), 0.90 (9H, dd, J = 5.8, 2.0 Hz); 13C NMR (Chloroform-d, 126 MHz) δ 172.4, 171.7, 155.9, 131.7, 118.6, 79.7, 65.8, 59.9, 50.8, 41.4, 30.9, 28.3, 24.8, 22.8, 21.9, 19.2, 18.0; HRMS (ESI) calculated for C19H34O5N2Na+ [M + Na]+ 393.2361, found 393.2360.
Tripeptide (23): Trifluoroacetic acid (5 mL) was added to a solution of compound S5 (40.42 mg, 2.15 mmol) in dry DCM (10 mL) at 0 °C. The reaction mixture was stirred at room temperature for 2 h. Volatiles were removed in vacuo and the residue was dried under high vacuum for 2 h to afford the corresponding amine, which was used in the next step without any further purification.
DIPEA (1.50 mL, 8.60 mmol) and HOAt (0.44 g, 3.23 mmol) were added to the above amino acid 9 (0.82 g, 2.15 mmol) solution in DCM (30 mL) at 0 °C and the mixture was stirred for 5 min. HATU (1.23 g, 3.23 mmol) was added and the resulting homogeneous reaction mixture was warmed to room temperature. After 12 h, the reaction mixture was cooled to 0 °C using an ice bath and quenched by the addition of H2O (10 mL). The mixture was transferred with H2O (10 mL) to a separatory funnel and extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with aqueous 1 N HCl (2 × 20 mL), brine (2 × 20 mL), H2O (20 mL), and brine (20 mL), dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography on silica gel, eluting with EtOAc/hexanes to afford tripeptide 23 in 91% yield over two steps (1.24 g, 1.96 mmol) as a white amorphous solid: Rf = 0.40 (ethyl acetate/hexanes = 1:1); = +17.10 (c 1.0, MeOH); 1H NMR (Methanol-d4, 500 MHz) δ 7.59 (1H, d, J = 1.9 Hz), 7.28 (1H, d, J = 8.5 Hz), 7.14 (1H, s), 7.04 (1H, dd, J = 8.7, 2.0 Hz), 5.91 (1H, ddt, J = 17.3, 10.8, 5.6 Hz), 5.31 (1H, dq, J = 17.3, 1.7 Hz), 5.25–5.18 (1H, m), 4.57 (2H, d, J = 5.7 Hz), 4.49–4.37 (2H, m), 4.15 (1H, t, J = 6.0 Hz), 4.13–4.00 (2H, m), 3.18 (1H, dd, J = 14.2, 8.3 Hz), 3.04 (1H, dd, J = 14.2, 6.9 Hz), 2.09–1.95 (1H, m), 1.81–1.68 (2H, m), 1.60 (1H, td, J = 8.4, 3.5 Hz), 0.95 (5H, d, J = 6.3 Hz), 0.89 (3H, d, J = 6.3 Hz), 0.66 (3H, d, J = 6.8 Hz), 0.60 (3H, d, J = 6.8 Hz), 0.01 (9H, s); 13C NMR (Methanol-d4, 126 MHz) δ 174.9, 173.7, 173.5, 158.6, 136.4, 133.3, 129.9, 126.5, 125.6, 122.5, 119.0, 118.7, 113.5, 110.7, 66.6, 64.3, 59.7, 57.5, 52.3, 41.1, 31.0, 28.6, 25.8, 23.4, 21.7, 19.4, 18.6, 17.8, −1.5; HRMS (ESI) calculated for C31H47O6N4ClSiNa+[M + Na]+ 657.2845, found 657.2846.
Hexapeptide (24): To a solution of allyl ester 23 (202.88 mg, 0.32 mmol) in anhydrous THF (10 mL) was added Pd(PPh3)4 (18.49 mg, 0.016 mmol) followed by the dropwise addition of morpholine (0.28 mL, 3.2 mmol), and the mixture was stirred for 45 min at room temperature. The reaction mixture was concentrated, and the residue was dissolved in DCM (30 mL). The resulting solution was washed with 1 N HCl (10 mL) and H2O (10 mL). The organic layer was dried with anhydrous Na2SO4 and concentrated. The crude acid intermediate S7 was obtained as an oil which was used in the next step without further purification.
Tripeptide 21 (268.29 mg, 0.32 mmol) was added to a 25 mL round-bottom flask equipped with a Teflon stir bar and fitted with a septum under an atmosphere of Ar. DCM (10 mL) was added via syringe and the mixture was stirred until it became homogeneous and colorless. The solution was cooled to 0 °C using an ice bath, and DBU (47.81 μL, 0.32 mmol) was added via syringe. The reaction mixture was stirred for 1.5 h at 0 °C. After the starting material was consumed completely as determined by TLC, HOAt (65.33 mg, 0.48 mmol) was added and the mixture was stirred for 5 min at 0 °C using an ice bath. S7 (190.16 mg, 0.32 mmol) and HATU (182.51 mg, 0.48 mmol) were added and the resulting homogeneous yellow reaction mixture was warmed to room temperature. After 12 h, the reaction mixture was cooled to 0 °C using an ice bath and quenched by the addition of H2O (5 mL). The mixture was transferred with H2O (5 mL) to a separatory funnel and extracted with EtOAc (2 × 30 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography on silica gel, eluting with EtOAc/hexanes to afford hexapeptide 24 in 83% yield over two steps (0.32 g, 0.27 mmol) as a white amorphous solid: Rf = 0.45 (methanol/dichloromethane = 1:20); = −6.30 (c 1.0, MeOH:DCM = 1:1); 1H NMR (DMSO-d6, 400 MHz) δ 11.04 (1H, d, J = 6.6 Hz), 8.13 (2H, dd, J = 9.2, 4.0 Hz), 8.06 (1H, d, J = 8.1 Hz), 7.86 (1H, d, J = 8.3 Hz), 7.66 (2H, d, J = 16.9 Hz), 7.31 (1H, d, J = 8.6 Hz), 7.23 (1H, d, J = 2.3 Hz), 7.13 (1H, d, J = 8.0 Hz), 7.01 (1H, dd, J = 8.6, 2.1 Hz), 6.71 (1H, t, J = 5.7 Hz), 6.00 (1H, t, J = 5.7 Hz), 5.85 (1H, ddt, J = 16.2, 10.7, 5.4 Hz), 5.36–5.24 (1H, m), 5.19 (1H, d, J = 10.5 Hz), 4.78 (1H, dd, J = 9.1, 3.1 Hz), 4.60 (1H, dd, J = 6.3, 3.1 Hz), 4.51 (2H, q, J = 8.2, 6.8 Hz), 4.35 (4H, dtd, J = 17.6, 7.5, 3.3 Hz), 4.13 (3H, q, J = 8.7 Hz), 3.91 (2H, dd, J = 9.9, 6.7 Hz), 3.01 (1H, dd, J = 14.4, 5.2 Hz), 2.86 (3H, q, J = 8.4, 7.9 Hz), 1.99 (1H, p, J = 6.8 Hz), 1.79 (1H, p, J = 6.6 Hz), 1.57 (3H, dq, J = 19.5, 13.5, 10.0 Hz), 1.35 (14H, s), 1.08–1.00 (1H, m), 0.94 (2H, t, J = 8.5 Hz), 0.85–0.69 (19H, m), 0.00 (9H, s), -0.05 (9H, s); 13C NMR (DMSO-d6, 101 MHz) δ 172.2, 172.0, 171.9, 170.7, 170.5, 169.0, 168.4, 156.1, 155.6, 134.6, 132.2, 128.4, 126.0, 123.0, 120.7, 118.0, 117.9, 112.7, 110.0, 77.4, 70.4, 65.1, 63.2, 61.8, 57.7, 55.4, 54.8, 51.9, 51.5, 42.5, 36.9, 29.9, 29.7, 29.0, 28.3, 27.8, 25.8, 25.8, 24.1, 22.9, 21.4, 19.2, 17.8, 17.3, 16.8, 14.3, 11.6, −1.6; HRMS (ESI) calculated for C56H93O14N8ClSi2Na+ [M + Na]+ 1215.5923, found 1215.5931.
Hexapeptide (S9): 1.0 M TBAF in THF (0.62 mL, 0.62 mmol) was added to a solution of the hexapeptide 24 (74.0 mg, 0.062 mmol) in THF (10 mL). The reaction mixture was stirred for 1 h at 50 °C. The reaction mixture was then cooled to 0 °C using an ice bath and quenched by the addition of a saturated aqueous solution of NH4Cl (5 mL). The mixture was transferred to a separatory funnel and extracted with EtOAc (3 × 30 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The crude product was used in the next step without further purification.
To a solution of the above crude product in a mixture of DCM (60 mL) and DMF (3 mL) were sequentially added DMAP (1.51 mg, 0.012 mmol), HOAt (12.66 mg, 0.093 mmol) and EDCI (17.83 mg, 0.093 mmol). The reaction mixture was stirred for 48 h at 30 °C. The reaction mixture was then cooled to 0 °C using an ice bath and quenched by the addition of a saturated aqueous solution of NH4Cl (20 mL). The mixture was transferred to a separatory funnel and extracted with DCM (3 × 60 mL). The combined organic layers were washed with H2O (2 × 30 mL) and brine (30 mL), dried over Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel, eluting with MeOH/DCM to afford hexapeptide S9 in 62% yield over two steps (35.77 mg, 0.04 mmol) as a white amorphous solid: Rf = 0.30 (methanol/dichloromethane = 1:20); = −7.10 (c 1.0, MeOH); 1H NMR (DMSO-d6, 500 MHz) δ 11.03 (1H, d, J = 2.5 Hz), 8.17 (1H, d, J = 6.4 Hz), 8.14 (1H, d, J = 7.3 Hz), 7.95 (1H, d, J = 7.4 Hz), 7.83 (1H, d, J = 8.2 Hz), 7.73 (1H, d, J = 8.7 Hz), 7.57 (1H, d, J = 2.0 Hz), 7.40 (1H, d, J = 7.0 Hz), 7.32 (1H, d, J = 8.7 Hz), 7.16 (1H, d, J = 2.5 Hz), 7.04 (1H, dd, J = 8.6, 2.1 Hz), 6.79 (1H, t, J = 5.8 Hz), 5.87 (1H, ddt, J = 17.4, 10.7, 5.4 Hz), 5.29 ( 1H, dq, J = 17.2, 1.7 Hz), 5.21–5.15 (1H, m), 4.65 (1H, dd, J = 8.6, 4.4 Hz), 4.62–4.47 (4H, m), 4.30–4.20 (2H, m), 4.00 (1H, q, J = 6.9 Hz), 3.79 (1H, t, J = 7.0 Hz), 3.04 (1H, dd, J = 14.3, 7.8 Hz), 2.91 (3H, dq, J = 17.2, 6.7 Hz), 1.88 (2H, dqt, J = 11.0, 6.9, 4.4 Hz), 1.81–1.71 (1H, m), 1.62–1.52 (3H, m), 1.48 (1H, dt, J = 13.4, 6.8 Hz), 1.36 (10H, s), 1.30 (2H, dt, J = 11.1, 5.8 Hz), 1.14 (1H, dp, J = 14.6, 7.4 Hz), 0.89–0.80 (12H, m), 0.63 (6H, t, J = 7.5 Hz); 13C NMR (DMSO-d6, 126 MHz) δ 172.0, 171.3, 171.2, 171.2, 171.1, 170.5, 168.7, 155.6, 134.6, 132.4, 128.4, 125.6, 123.0, 120.8, 117.8, 117.6, 112.8, 109.6, 77.4, 70.1, 64.9, 59.7, 56.5, 56.0, 55.3, 54.0, 53.2, 51.7, 36.3, 29.2, 28.3, 27.5, 26.8, 26.2, 25.7, 24.3, 22.7, 22.1, 18.8, 18.5, 18.0, 14.5, 11.4; HRMS (ESI) calculated for C45H67O11N8ClNa+ [M + Na]+ 953.4507, found 953.4510.
Nicrophorusamide A (25): Cyclic hexapeptide S9 (46.52 mg, 0.05 mmol) was dissolved in a saturated methanolic NH3 solution (10 mL) and stirred at 0 °C. After the starting material was consumed completely as determined by TLC, the reaction was concentrated by rotary evaporation. The residue was azeotropically dried by concentration from methanol (2 × 10 mL) and then used in the next step without further purification.
To a solution of the above amide in dry DCM (5 mL) was added trifluoroacetic acid (2 mL) at 0 °C. The reaction mixture was stirred at ambient temperature for 0.5 h. Volatiles were removed in vacuo and the residue was purified by column chromatography on silica gel, eluting with MeOH/DCM to afford 25 in 55% yield (21.71 mg, 0.0275 mmol) as a white amorphous solid: Rf = 0.27 (methanol/dichloromethane = 1:2); = −15.50 (c 0.2, MeOH); IR (MeOH) 3441, 3385, 2964, 1676, 1631, 1535, 1201, 1055 cm−1; 1H NMR (DMSO-d6, 500 MHz) δ 11.05 (d, J = 2.4 Hz, 1H), 8.48 (d, J = 6.0 Hz, 1H), 7.97 (d, J = 7.9 Hz, 1H), 7.85 (dd, J = 7.5, 4.8 Hz, 2H), 7.76 (d, J = 6.1 Hz, 3H), 7.60–7.55 (m, 2H), 7.48 (d, J = 7.3 Hz, 1H), 7.33 (d, J = 8.6 Hz, 2H), 7.28 (d, J = 2.7 Hz, 1H), 7.15 (d, J = 2.3 Hz, 1H), 7.04 (dd, J = 8.5, 2.1 Hz, 1H), 5.91 (d, J = 6.0 Hz, 1H), 4.65–4.56 (m, 2H), 4.47 (d, J = 3.8 Hz, 1H), 4.24 (ddt, J = 9.7, 7.3, 4.0 Hz, 2H), 3.99 (q, J = 6.7 Hz, 1H), 3.75 (t, J = 7.3 Hz, 1H), 3.11 (dd, J = 14.2, 7.3 Hz, 1H), 2.89 (dd, J = 14.2, 7.0 Hz, 1H), 2.81 (t, J = 6.6 Hz, 2H), 1.86 (dh, J = 28.2, 6.8 Hz, 3H), 1.70–1.59 (m, 1H), 1.58–1.54 (m, 2H), 1.54–1.48 (m, 2H), 1.34 (dq, J = 13.8, 6.8 Hz, 1H), 1.15 (dp, J = 14.8, 7.4 Hz, 1H), 0.89–0.75 (m, 13H), 0.70 (dd, J = 10.9, 6.7 Hz, 6H); 13C NMR (DMSO-d6, 126 MHz) δ 173.2, 172.1, 171.7, 171.1, 170.6, 170.4, 169.5, 134.5, 128.4, 125.5, 123.0, 120.7, 117.5, 112.7, 109.8, 70.8, 60.4, 56.6, 55.7, 54.8, 53.5, 53.2, 51.5, 38.4, 36.2, 29.1, 26.8, 26.7, 25.6, 24.3, 23.8, 22.7, 21.8, 18.8, 18.4, 14.5, 11.3; HRMS (ESI) calculated for C37H57O8N9Cl+ [M + H]+ 790.4009, found 790.4013.