The preferred method of detecting and assaying enzymes is to incubate with a substance which is as close to the natural substrate as possible, but which will liberate a molecule that can be quantified on the basis of its colour, fluorescence, or chemiluminescence. The last two properties are particularly sensitive, but their measurement requires specialised equipment. The most widely used substrates for the assay of glycosides are the 4-and 2nitrophenyl glycosides, but the nitrophenols released, after basification, have weak yellow colours (A,, -410 nm) and endogenous coloured species may interfere in the assays.
Appropriate derivatives of the highly coloured 2-methoxy-4-(2nitrovinyl)phenol' (2a; Amax 505, E 27 000) are good substrates for the calorimetric assay of glycosidas-es2, phosphatases, and esterases3. However, the colour of the phenol released tends to fade under alkaline conditions~ and the substrates are not as soluble in water as is required for general use.
The condensation of vanillyl2-acetamido-2-deoxy-/I-D-glucopyranoside (lb) and vanillyl j?-D-galactopyranoside (lc) with various heterocycles having either an active methyl or methylene group has now been studied in order to enhance the colour4 of the released phenol and the solubility of the substrate. The resulting substrates are suitable for the assay of 2-acetamido-2-deoxy-~-D-glucopyranosidase (NAG) and j?-D-galactopyranosidase, respectively.
Condensation of lb with an equimolar amount of rhodanine-3-acetic acid in the presence of ammonia and ammonium chloride afforded 93% of ammonium 5-[4-(2-acetamido-2-deoxy-~-~-glucopyranosyloxy)-3-methoxyphenylmethylene]-2-thioxot~a-zoli~n~-one-3-acetate (3b, m-p. 165-173" (dec.), [or&, + 18" (methyl sulphoxide)j as a bright yellow monohydrate. Although the reaction mixture was heterogeneous throughout (4 h at 60" in ethanol), the product contained (h.p.1.c.) eO.5% of lb.