A mixture of AT-125 (178 mg; 1 mmole), 9 fluorenylmethylchloroformate (285 mg, 1.1 mmole), and potassium bicarbonate (370 mg; 3.7 mmole) in 5 ml of water was stirred for 20 hours at room temperature. The gelatinous mixture is acidified to pH 2 with hydrochloric acid, evaporated to dryness under reduced pressure, and extracted with acetone. The acetone extracts are adsorbed onto silica gel and chromatographed on 40 g of silica gel with methylene chloride:isopropanol:acetic acid (100:3:1). Fractions of 20 ml are collected and monitored by TLC (U.S. visualization). The first 8 fractions contain components derived from the chloroformate reagent only including 9-fluorenylmethyl alcohol. Fractions 11-17 contain 206 mg of chromatographically homogeneous 3-chloro-α-[[(9H-fluoren-9-yl-methoxy)carbonyl]amino]-4,5-dihydro-5-isoxazoleacetic acid, and fractions 21-50 contain 132 mg of chromatographically homogeneous 3-chloro-α-[[(3-chloro-4,5-dihydro-5-isoxazolyl)[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino]-4,5-dihydro-5-isoxazoleacetic acid. Crystallization of fractions 11-17 from aqueous methanol and recrystallization from acetone-cyclohexane yields 3-chloro-α-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-4,5-dihydro-5-isoxazoleacetic acid m.p. 175°-176°.
