Diisopropylethylamine (17.2microl., 0.1mmol) was added to (2S, 4E)-8-^tbutyldimethylsilyloxy-2-methyloct-4-enoic acid (18) in dry dichloromethane (0.5ml) under argon. BOP-Cl (12.7mg, 0.05mmol) was then added and the mixture stirred for 1h. Crude L-alanyl-L-alanyl-L-alanine methyl ester (7) (16.2mg, 0.05mmol) in dry dichloromethane (0.2ml) was then added and the reaction stirred at room temperature for 3 days. Water was added and the reaction mixture acidified to pH 1 with 2M hydrochloric acid. The crude product was extracted with ethyl acetate (5x5ml), washed with brine (5ml), dried and finally concentrated under reduced pressure. Recrystallisation of the crude product from ethyl acetate afforded the coupled adduct (19) as a white solid (18mg, 77%), m.p. = 171-2 degC, [alpha]D = -12deg (c = 1.0, EtOAc).

Proton nmr (300 MHz, CDCl3) delta 7.05 (1H, br.d, J = 7.4 Hz, NH), 6.88 (1H, br.d, J = 7.4 Hz, NH), 6.33 (1H, br.d, J = 7.4 Hz, NH), 5.49-5.36 (2H, m, CH=CH), 4.56-4.44 (3H, m, 3xNHCH), 3.72 (3H, s, CO2CH3),3.56 (2H, t, J = 6.5 Hz, CH2OSi), 2.52-2.45 (1H, m, CHaHbCHCH3), 2.21-2.15 (1H, m, CHaHbCHCH3), 2.17-1.95 (3H,m, CH2CH=CH, CH2CHCONH), 1.57-1.52 (2H, m, -CH2-), 1.41-1.32 (9H, m, 3xNHCHCH3), 1.10 (3H, d, J = 6.5 Hz, CH3CONH), 0.86 (9H, s, C(CH3)3), 0.01 (6H, s, Si(CH3)2); Vmax (CHCl3) 3279 br, 2929, 2856, 1631 cm-¹; m/z (FAB spectrum, +ve ion) 514 (80 %) (M+H)⁺, 456 (40 %), 411, 340 (35 %), 312, 255, 211, 175 (100 %), 133. Found (M+H)⁺ 514.3297. C25H48O6N3 requires 514.3312.

Preparation of (2S, 4E)-N-[8'-Hydroxy-2-methyloct-4-enoyl]-L-alanyl-L-alanyl-L-alanyl 1,18-lactone (20)

To a stirred solution of the ester (19) (18mg, 0.04mmol) in ethanol (0.5ml) was added an aqueous solution of 10M potassium hydroxide (10microl.). When the reaction had gone to completion by tlc the mixture was acidified to pH 1 with 2M hydrochloric acid, then stirred at room temperature for 8h., after which the ethanol removed under reduced pressure. The intermediate seco acid was then extracted with ethyl acetate (5x5ml) and the organic extracts combined and washed with brine (5ml). After drying the organics were concentrated to give the crude seco acid which was then dissolved in dry dichloromethane / N,N-dimethylformamide (1ml, 1:1 v/v) under an inert atmosphere. Diisopropylethylamine (14microl., 0.08mmol) was added and the mixture stirred for 30min. BOP-Cl (10mg, 0.04mmol) was then added and the reaction mixture stirred for 3 days. Water (5ml) was added and the reaction mixture acidified to pH 1 with 2M hydrochloric acid. The crude product was extracted with ethyl acetate (5x5ml), washed with brine (5ml), dried and finally concentrated to afford the macrocycle (20) as a pale yellow oil (8mg, 58% ).

Proton nmr (300 MHz, CDCl3) delta 7.85 (1H, br.d, J = 7.4 Hz, NH), 7.50 (1H, br.d, J = 6.4 Hz, NH), 7.17 (1H, br.d, J = 7.4 Hz, NH), 5.50-5.31 (2H, m, CH=CH), 4.56-4.40 (3H, m, 3xNHCH), 3.61 (2H, t, J = 5.4 Hz, CH2OCO), 2.58-2.44 (1H, m, CHaHbCHCH3), 2.35-2.21 (3H, m, CHaHbCHCH3, CH2CH=CH), 2.10-2.01 (1H, m, CH2CHCONH), 1.56-1.50 (2H, m, -CH2-), 1.40-1.31 (9H, m, 3xNHCHCH3), 1.10 (3H, d, J = 6.5 Hz, CH3CHCONH); Vmax (CHCl3) 3350 br, 2927, 2854, 1651, 1454 cm^-1; m/z (FAB spectrum, +ve ion) 368 (20 %) (M+H)⁺, 354 (70 %), 340, 326 (100 %), 312, 298, 281, 267, 255 (80 %).