Glycosides Having Chromophores as Substrates for Sensitive Enzyme Analysis. V. Synthesis of 6'-O-Substituted 2'-7'-Dichlorofluorescein N-Acetyl-β-D-glucosaminides as Substrates for the Rate-Assay of N-Acetyl-β-D-glucosaminidase

Abstract

Sixteen novel 6'-O-substituted 2', 7'-dichlorofluorescein N-acetyl-β-D-glucosaminides (2a-p) were synthesized from 2', 7'-dichlorofluorescein 2, 3, 4, 6-tetraacetyl-β-D-glucosaminide (10), prepared through 3 steps from 2', 7'-dichlorofluorescein (3). These N-acetyl-β-D-glucosaminides were examined to evaluate their solubility under the weakly acidic rate-assay conditions (pH 5.0) and their kinetic parameters with N-acetyl-β-D-glucosaminidase. Among these compounds, 6'-O-carboxymethyl- (2a), 6'-O-(N, N-dimethylaminoethyl)- (2i), and 6'-O-sulfoxyethyl-2', 7'-dichlorofluorescein N-acetyl-β-D-glucosaminides (2p), are considered to be potential chromogenic substrates for the rate-assay of N-acetyl-β-D-glucosaminidase, since they showed good solubility of more than 10mM, moderate K_m values of 1.46-2.04mM, and higher sensitivity to the enzyme than 2-chloro-4-nitrophenyl-N-acetyl-β-D-glucosaminide.