X-GlcNAc (5-Bromo-4-chloro-3-indolyl N-acetyl-β-D-glucosaminide, CAS 4264-82-8) is a chromogenic substrate specifically designed for detecting β-N-acetylglucosaminidase (β-NAG, EC 3.2.1.52) activity in biochemical assays, microbial screening, and histochemical studies of lysosomal storage disorders and glycosaminoglycan metabolism. Hydrolysis by β-NAG cleaves the β-glycosidic bond between the indolyl aglycone and N-acetylglucosamine moiety, releasing 5-bromo-4-chloroindoxyl, which spontaneously dimerizes and oxidizes to form an insoluble blue precipitate for precise enzyme localization without diffusion artifacts. This substrate excels in screening bacterial chitinases/lysozymes, diagnosing mucopolysaccharidoses (Sanfilippo syndrome), monitoring kidney damage via urinary NAG, and environmental microbiology for cellulolytic fungi, offering superior spatial resolution and permanence compared to soluble p-nitrophenyl substrates with optimal activity at pH 4.5-5.5 and Km values of 0.2-0.8 mM.
Appearance
- Bright yellow to off-white crystalline powder, odorless.
- Soluble in DMF/DMSO (>20 mM); sparingly water-soluble (1-3 mM).
Source
- Koenigs-Knorr glycosylation: peracetylated GlcNAc-α-bromide + 5-Br-4-Cl-indol-3-ol, Ag2CO3 catalysis, followed by deacetylation.
- Commercial molecular biology grade from specialized enzyme substrate manufacturers.
Molecular Weight and Structure
- C16H18BrClN2O6, 449.68 g/mol; exact mass 449.9528 Da.
- 5-Br-4-Cl-3-indolyl β-D-GlcNAc; β-(1→O)-glycosidic linkage.
Sugar Specificity
- Highly selective for β-N-acetylglucosaminidases (GH3/GH20); Km 0.2-0.8 mM.
- Minimal β-GalNAc or β-glucosidase activity (10-50x lower rates).
Biological Activity
- β-NAG hydrolysis → indolyl + GlcNAc → insoluble blue precipitate.
- Optimal pH 4.5-5.5, 37°C; visible color development 30 min-4h.
Purity and Microbial Contamination
- ≥98% HPLC purity; β-anomer >97%; free indoxyl <0.1%.
- Endotoxin-free; sterile lyophilized powder.
Identity and Quality Control
- LC-MS (m/z 450.1 [M+H]+), 1H-NMR (β-H1 J=8.2 Hz), functional assay.
- TLC single spot (Rf 0.5, EtOAc:MeOH 3:1); CAS verified.
Shelf Life and Storage
- 3 years desiccated -20°C; amber vials, avoid moisture/light.
- DMF stocks stable 6 months -20°C; fresh aqueous dilutions.
Application
- Urinary β-NAG for kidney damage/diabetes monitoring.
- Bacterial chitinase/lysozyme screening; fungal cellulase assays.
- Mucopolysaccharidosis diagnostics; histochemical enzyme localization.
Key Characteristics
- Insoluble blue precipitate enables precise localization; no diffusion.
- High contrast vs. tissue; superior to pNP-GlcNAc for histology.
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