Abstract
Glycyrrhetylamino acids (5a-c) were prepared by the condensation of glycyrrhetic acid (GA) with amino acids (glycine, γ-aminobutyric acid, and ε-aminohexanoic acid), which were selected for use as chemical bridges between the hapten and carrier protein in an enzyme immunoassay (EIA) for GA. The condensation was carried out in the presence of dicyclohexylcarbodiimide (method A), diphenyl phosphorazidate (method B) or diethyl phosphorocyanidate (method C), and method C gave the desired glycyrrhetylamino acids (5a-c) in the best yields. β-Galactosidase was used as the labeled enzyme and was conjugated with GA by the N-hydroxysuccinimide ester method. Separation of bound and free fractions was performed by a double antibody method using a goat antiserum to rabbit IgG. 7-β-D-Galactopyranosyloxy-4-methylcoumarin was used as substrate for the fluorometric assay of β-galactosidase activity. A satisfactory standard curve for GA was obtained in the range of 2.5-250 ng/ml.
