Glycosides Having Chromophores as Substrates for Sensitive Enzyme Analysis. IV. Synthesis of N-Acetyl-β-D-glucosaminides of Fluorescein Derivatives and Their Application to the Rate-Assay of N-Acetyl-β-D-glucosaminidase

Abstract

Three novel N-acetyl-β-D-glucosaminides, 2', 7'-dichlorofluorescein mono(N-acetyl-β-D-glucosaminide) (6a), fluorescein mono(N-acetyl-β-D-glucosaminide) (6b) and 2', 7'-dichlorofluorescein di(N-acetyl-β-D-glucosaminide) (7a), were synthesized by the introduction of N-acetyl-β-D-glucosaminyl group into fluorescein derivatives followed by the removal of the protecting group. Compounds 6a, 6b and 7a were hydrolyzed by N-acetyl-β-D-glucosaminidase to give products showing high absorbance at a long absorption wavelength (500, 465 and 485 nm) under weakly acidic rate-assay conditions (pH 5.0). The K_m values for 6a and 7a were 0.56 and 0.86 mM, respectively. Among these compounds, 7a is considered to be the most potential chromogenic substrate for the rate-assay of N-acetyl-β-D-glucosaminidase, since it gives a clear color generation from colorless to orange color (λ_max 280→485 nm) by enzyme hydrolysis and has a higher water solubility of more than 30 mM.