A solution of (2'S, 3E)-4-(N-tert-butoxycarbonylpyrrolidin-2-yl)but-3-enoylglycyl-L-alanine ethyl ester (21) (180mg, 0.51mmol) in dichloromethane (2ml) was treated with trifluoroacetic acid (1ml) in dichloromethane (1ml) and the resulting solution stirred at room temperature for 1h. The solution was then concentrated to dryness in vacuo, and the residue dissolved in dry tetrahydrofuran (2ml) and placed under an argon atomsphere. N-Methylmorpholine (110microl, 1.04mmol) was then added, and the mixture stirred for 5min., before being added to a stirred solution of N-tert-butoxycarbonyl-L-alanine (110mg, 0.57mmol) in dry tetrahydrofuran (2ml) that had been pre-treated with N-methylmorpholine (60microl, 0.55mmol) and ethyl chloroformate (54microl, 0.57mmol) at 0degC for 10min. The resulting mixture was stirred at 0degC for 30min., then at room temperature for 5.5h. Ethyl acetate (30ml) was then added, and the solution washed sequentially with 1M hydrochloric acid (20ml), sodium bicarbonate (20ml), water (25ml), and saturated sodium chloride (25ml), then dried over magnesium sulphate and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (petroleum ether/ethyl acetate 1:2) to give (2'S, 3E)-4-(N-tert-butoxycarbonyl-L-alanylpyrrolidin-2-yl)but-3-enoylglycyl-L-lanine ethyl ester (22) (58mg, 27%) as a colourless oil, [alpha]D -24deg (c 1.0, dichloromethane).
Proton Nmr deltaH (300MHz, CDCl3) 1.18-1.44(18H, m, 2xCHCH3, CH2CH3, O(C(CH3)3), 1.72-2.07 (4H, m, 2xCH2), 2.96(2H, apparent d, J=7.0Hz, CH2C=C), 3.42-3.62(2H, m, CH2NBoc), 4.12-4.21(2H, m, OCH2CH3), 4.34-4.53(3H, m, 3xCHN), 5.35-5.41 (1H, br. m, NH), 548(1H, dd, J=6.0, 15.5Hz, CH=C), 5.67-5.54(1H, m, C=CH), 6.66 (1H, br. d, J=7.0Hz, NH); Vmax: 3305, 1740, 1710, 1643cm-1; m/z (NH3, CI) 426(M+H+, 100%). Found [M+H]+ 426.2603, C21H36N3O6 requires 426.2604.