A solution of N-^tbutoxycarbonyl-L-alanyl-L-alanyl-L-alanine methyl ester (7) (100mg, 0.41mmol) in dry dichloromethane (5ml) was stirred at 0degC under an argon atmosphere, then trifluoroacetic acid (5ml) was added and the mixture stirred at room temperature for 1h. The reaction mixture was then diluted with ethyl acetate (50ml), washed successively with aqueous saturated sodium bicarbonate and brine, dried, and the solvent removed under reduced pressure. The residue was dissolved in dry N,N-dimethylformamide (5ml), and treated sequentially with (2R/S, 6S, 8R/S)-8-^tbutyldimethylsilyloxy-2,6-dimethyl nonanoic acid (6) (130mg, 0.41mmol), N-hydroxy-benzotriazole (66mg, 0.49mmol), triethylamine (50mg, 0.49mmol), 4,4-dimethylaminopyridine (5mg), and finally N,N-dicyclohexylcarbodiimide (101mg, 0.49mmol) at 0degC under argon. After stirring at this temperature for 20min. a white precipitate of N,N-dicyclohexylurea was observed and the reaction mixture stirred at room temperature for a further 72 h. N,N-Dicyclohexylurea was removed by filtration through a Celite pad and the mixture diluted with ethyl acetate (50ml). The organics were washed successively with 1 M hydrochloric acid, water (3x30ml) and brine before being dried and concentrated to yield the crude product. Recrystallisation from ethyl acetate-cyclohexane gave the coupled product as a white solid (50mg, 24%), m.p. = 188-9degC.

Proton nmr (300 MHz, CDCl3) delta 6.87 (1H, br.d, J = 7.4 Hz, NH), 6.75 (1H, br.d, J = 6.4 Hz, NH), 6.15 (1H, br.d, J = 7.4 Hz, NH), 4.45-4.47 (3H, m, 3xNHCH), 3.84 (1H, m, CHOSi), 3.73 (3H, s, CO2CH3), 2.18 (1H, m, CH2CHCONH), 1.60 (1H, m, -CH-), 1.40-1.34 (12H, m, CH3CHOSi, 3xNHCHCH3), 1.30-1.18 (8H, m, 4xCH2), 1.08 (3H, m, CHCH3CONH), 0.85 (12H, m, CH3CH, C(CH3)3), 0.02 (6H, m, Si(CH3)2); Vmax (CDCl3) 3342 br, 3296 br, 2928, 2875, 1630 cm^-1; m/z (CI, NH3) 562 (M+NH4)⁺, 544 (M+H)⁺, 447, 430, 342, 256, 231, 201, 175, 160, 149, 132; m/z (FAB spectrum, +ve ion) 566 (M+Na)⁺, 544 (M+H)⁺, 528, 486, 441, 412, 370, 343, 309, 238, 175, 73. Found (M+H)⁺ 544.3785. C27H54O6N3Si requires 544.3782.

Preparation of (2R/S, 6S,8R/S)-N-[8-Hydroxy-2,6-dimethylnonanoyl]-L-alanyl-L-alanyl-L-alanyl-1,18-lactone(9)

To a stirred solution of the ester (8) (26mg, 0.07mmol) in ethanol (0.5ml) was added an aqueous 10 M solution of potassium hydroxide (20microl.). When the reaction had gone to completion by tlc the mixture was cooled to -10degC, and acidified with 2M hydrochloric acid to pH 1. The resulting mixture was stirred at room temperature for 8h., and then the ethanol was removed under reduced pressure. The intermediate seco acid was 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 immediately dissolved in dry dichloromethane / N,N-dimethylformamide (1ml, 1:1 v/v) under argon. Diisopropylethylamine (27microl., 0.15 mmol) was added and the mixture stirred for 30min. BOP-Cl (20mg, 0.08mmol) was then added and the reaction mixture stirred at room temperature for 3 days. Water was then 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 concentrated under reduced pressure. Recrystallisation of the crude product from ethyl acetate afforded the macrocycle (9) as a white solid (10mg, 48% overall).

Proton nmr (300 MHz, CDCl3) delta 6.80 (1H, br.d, J = 7.4 Hz, NH), 6.75 (1H, br.d, J = 6.4 Hz, NH), 6.16 (1H, br.d, J = 7.4 Hz, NH), 4.62-4.40 (3H, m, 3xNHCH), 3.90 (1H, m, CHOSi), 2.25 (1H, m, CH2CHCONH), 1.64 (1H, m, -CH-), 1.49-1.35 (12H, m, CH3CHO, 3xNHCHCH3), 1.30-1.15 (8H, m, 4xCH2), 0.82 (6H, m, 2xCH3CH,); Vmax (CHCl3) 3583-3100 br, 2927, 2854, 1688, 1657, 1634, 1575 cm^-1; m/z (CI, NH3) 415 (M+NH4)⁺, 398 (M+H)⁺, 386, 344, 328, 274, 256, 220, 202, 168, 133; m/z (FAB spectrum, +ve ion) 398 (35 %) (M+H)⁺, 327, 256, 238, 161, 130 (80 %). Found (M+H)⁺ 398.2678. C20H36O6N3 requires 398.2655.