Reactions of DNP-Amino Acids with Hydroxylamine

Regeneration of free amino acids from dinitrophenylated (DNP) amino acids was carried out by the use of hydroxylamine as the nucleophilic reagent. For this reaction, ca. 1 mg of DNP-amino acid is dissolved in 1 drop of 2-methoxyethanol, 1 drop each of 20% NH2OH·HCl and 20% KOH are added, and the mixture is shaken for a few minutes at room temperature. One drop each of 20% AcOH and acetone are then added, and the mixture is submitted to thin-layer chromatography for the detection free amino acids. Good results were obtained with dinitrophenylated glycine, L-alanine, L-leucine, L-valine, L-phenylalanine, L-proline, L-serine, DL-threonine, L-tyrosine, L-glutamic acid, L-aspartic acid, L-cysteine, DL-methionine, L-lysine, and L-arginine. In the reaction of DNP-DL-methionine, a minute amount of Ninhydrin-positive substance was produced by the action of NH2OH on methionine. A good result was not obtained with DNP-L-histidine. This reaction requires an excess of alkali hydroxide and a good result is obtained when the molar ratio of NH2OH·HCl to KOH is 1 : 2. This is not merely a substitution reaction of hydroxylamine and amino acid but, considering the formation of 3-amino-4-nitro-benzoquinone 1, 2-dioxime (II), substitution of -NHCH2COOH with -NHOH is followed by dehydrative cyclization of -NHOH with the neighboring -NO2 to form a benzofuroxane derivative (VII) which undergoes reduction to an o-quinone dioxime derivative, and introduction of an NH2 group into the 3-position at about the same time.