Sodium Bicarbonate → O-Benzoylhydroxylamine

To a solution of O-Benzoylhydroxylamine hydrochloride (85 mg, 0.49 mmol, prepared as described in the procedure of Carpino et al., 1959) in water (5 ml) was added at room temperature a 1 M solution of sodium bicarbonate until the effervescence ended. The organic material was extracted three times with CH2Cl2 (10 ml per extraction). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to give O-Benzoylhydroxylamine (49 mg, 73%). In a second flask Z-Ala-Ala-Asn-OH (19) (130 mg, 0.318 mmol) was dissolved in a mixture of dry THF (5 ml) and dry DMF (3 ml). To this stirred solution were added isobutyl chloroformate (41 μl, 0.318 mmol) and NMM (35 μl, 0.318 mmol) at −15° C. After stirring for 15 minutes O-Benzoylhydroxylamine in dry THF (2 ml) was added and the mixture was stirred 14 h, during which time it was allowed to warm to room temperature. The solvent was evaporated in vacuo and the obtained residue was washed with cold KHSO4 (5% in water, 5 ml). The precipitate was dissolved in ethyl acetate (10 ml), washed with water (3×5 ml) and dried over Na2SO4. After filtration the solvent was evaporated under reduced pressure. The crude compound was purified by preparative HPLC to give the product as an oil (77 mg, 46%).—TLC (MeOH/CHCl3, 1:9): Rf=0.31.—1H NMR (500 MHz, DMSO-d6): δ=1.19 (d, 3 H, J=6.9 Hz, CH3), 1.21 (d, 3 H, J=7.0 Hz, CH3), 2.78-2.96 (m, 2 H, CHCH2), 4.04-4.08 (m, 1 H, CH), 4.25-4.34 (m, 1 H, CH), 4.48-4.55 (m, 1 H, CH), 4.97-5.04 (m, 2 H, CH2O), 7.26-7.42 (m, 10 H, aryl-H), 7.93 (d, 2 H, J=8.2 Hz, NH).—MS (EI) m/z (%): 528 [M+H+], 550 [M+Na+], 566 [M+K+].