Chloroacetic Acid → O-(2-diazo-3-hydroxy-1-oxopropyl)-L-serine

To a solution of O-(L-serinyl)-N-trifluoroacetyl-L-serine (400 mg) and 4M aqueous chloroacetic acid (50 μl) is water (4 ml) was added sodium nitrite (193 mg) at room temperature. After stirring for 15 minutes at room temperature, the reaction mixture was neutrized with 2M tris buffer solution to pH 7.0 to 7.3 and then Acylase I (acylase, Sigma A-7264, made by Sigma Chemicals) (60 mg) was added. The mixture was stirred for 5 hours at room temperature, and chromatographed on carbon (50 ml) eluting successively with water and a 9:1 mixture of water and acetone to give a crude product, which was chromatographed on silica gel eluting with a mixture of methylene chloride, methanol and water (10:6:1 to 5:3:1), and also chromatographed on silica gel eluting with a 5:3:1 mixture of methylene chloride, methanol and water (5:3:1) to give a pure product which was lyophylized to give O-(2-diazo-3-hydroxy-1-oxopropyl)-L-serine (33 mg).