Enzymatic synthesis of N-glucosyluronic acid conjugates.

Chemicals-o-Aminophenyl glucosiduronic acid was kindly supplied by Dr. R. T. Williams, St. Mary’s Hospital Medical School, London. oAminobenzoyl and p-aminobenzoyl glucosiduronic acids were prepared enzymatically with UDP-glucuronic acid’ and aglycone (5). Phenolphthalein glucosiduronic acid and bacterial P-glucuronidase were obtained from the Sigma Chemical Company, St. Louis, Missouri. UDP-glucuronic acid was prepared enzymatically from UDPG, with UDPG dehydrogenase and DPN (4). An aniline-glucuronic acid complex was generously supplied by Dr. R. T. Williams (6). This compound contained 2 moles of aniline per mole of glucuronic acid. 1 mole of aniline could be removed by washing a solution of the compound at pH 8.0 with ether. The resulting aniline-glucuronic acid compound did not reduce triphenyltetrazolium in 0.5 N NaOH, but, following acidification with 0.1 N HCl, both the reducing group of glucuronic acid and the amino group of aniline were liberated. These observations indicated that the compound was N-phenylglucuronosylamine, in which the amino group was linked to carbon 1 of glucuronic acid. Methods--Aniline and p-toluidine were extracted at pH 7.0 or above into 5 volumes of ethylene dichloride containing 1.5 per cent isoamyl alcohol. The compounds were returned to 1 ml. of 0.1 N HCl and determined in 0.5 ml. of the acid extract by diazotization and by coupling