Chemical and Pharmaceutical Bulletin
Online ISSN : 1347-5223
Print ISSN : 0009-2363
Synthesis of 4-Aminophenyl N-Acetyl-β-D-glucosaminide Derivatives and Their Application to the Rate-Assay of N-Acetyl-β-D-glucosaminidase
Kouichi KASAIKiyoshi OKADANobuyuki YAMAJI
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1995 Volume 43 Issue 2 Pages 266-270


Four N-acetyl-β-D-glucosaminides, 4-amino-2, 6-dibromophenyl (1a), 4-amino-2, 6-dichlorophenyl (1b), 4-amino-2-chlorophenyl (1c) and 4-aminophenyl N-acetyl-β-D-glucosaminides (1d) were synthesized. Substrates 1a-c were hydrolyzed by N-acetyl-β-D-glucosaminidase and the released aglycones reacted with N-ethyl-N-(3-methylphenyl)-N'-succinylethylenediamine to produce indoaniline dyes in the presence of bilirubin oxidase under weakly acidic rate-assay conditions (pH 5.0). The Km values for 1a-c were 1.97, 1.65 and 1.39 mM, respectively. Among these compounds, 1b is considered to be the substrate with most potential for the rate-assay of N-acetyl-β-D-glucosaminidase, since it showed the largest Vmax value and the strongest color generation from colorless to green (λmax 302→718 nm)following enzyme hydrolysis and the coupling reaction. Furthermore, 1b was moderately soluble and stable in aqueous solution and exhibited about a 5.9-fold higher sensitivity to the enzyme than 2-chloro-4-nitrophenyl N-acetyl-β-D-glucosaminide.

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