5-Bromo-4-chloro-3-indolyl-N-acetyl-beta-D-glucosaminide
[CAS# 4264-82-8]

Identification

• Classification: Biochemical >> Carbohydrate >> Monosaccharide
• Name: 5-Bromo-4-chloro-3-indolyl-N-acetyl-beta-D-glucosaminide
• Synonyms: X-Glucosaminide
• Molecular Formula: C₁₆H₁₈BrClN₂O₆
• Molecular Weight: 449.68
• CAS Registry Number: 4264-82-8
• EC Number: 805-331-1
• SMILES: CC(=O)N[C@@H]1[C@H]([C@@H]([C@H](O[C@H]1OC2=CNC3=C2C(=C(C=C3)Br)Cl)CO)O)O

Properties

• Density: 1.8±0.1 g/cm³ Calc.^*
• Boiling point: 777.5±60.0 ºC 760 mmHg (Calc.)^*
• Flash point: 424.0±32.9 ºC (Calc.)^*
• Index of refraction: 1.702 (Calc.)^*

^* Calculated using Advanced Chemistry Development (ACD/Labs) Software.

Safety Data

• Hazard Symbols: GHS07 Warning
• Hazard Statements: H315-H319-H335
• Precautionary Statements: P261-P264-P271-P280-P302+P352-P304+P340+P312-P305+P351+P338-P332+P313-P337+P313-P362-P403+P233-P405-P501

Discovory and Applicatios

5-Bromo-4-chloro-3-indolyl-N-acetyl-β-D-glucosaminide is a synthetic chemical substrate designed for the specific detection of β-N-acetylglucosaminidase activity in biological and analytical systems. The compound belongs to a class of chromogenic glycosidase substrates based on substituted indole derivatives. Its structure consists of a substituted indolyl core with bromine and chlorine atoms at the 5- and 4-positions, respectively, and a β-linked N-acetyl-D-glucosaminide group attached to the nitrogen atom of the indole ring. The combination of halogen substituents and the glycosidic linkage confers both chemical stability and distinct chromogenic properties upon enzymatic cleavage.

The development of this compound was part of broader research efforts in the mid-to-late 20th century to create chromogenic substrates for enzymatic detection. Earlier efforts focused on β-galactosidase substrates, such as 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, which became widely used in microbiological and molecular biology assays. Following the success of such substrates, researchers synthesized analogs for other glycosidases. The compound 5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-glucosaminide was introduced to enable the visual detection of β-N-acetylglucosaminidase activity using a similar principle.

In the presence of β-N-acetylglucosaminidase, the substrate undergoes enzymatic hydrolysis, releasing the substituted indoxyl moiety from the N-acetylglucosamine group. This liberated indoxyl compound is then oxidized in situ to form a blue-colored dimeric product, specifically 5,5'-dibromo-4,4'-dichloro-indigo. The formation of this insoluble blue precipitate marks the site of enzymatic activity and can be visualized either macroscopically or microscopically. This property makes the compound highly useful in histochemical applications for localizing enzyme activity in tissues or cell cultures.

One major application of 5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-glucosaminide is in lysosomal enzyme studies. The compound is used to detect the presence and activity of β-N-acetylglucosaminidase in human and animal tissues. In histology, frozen or fixed tissue sections are incubated with the substrate under controlled pH and temperature conditions. Regions with active enzyme produce a blue precipitate, allowing researchers to examine the distribution of lysosomal activity. This method is employed in pathology to identify enzyme deficiencies in lysosomal storage disorders and other metabolic diseases.

In addition to histochemistry, the substrate is employed in microbiological assays. It is used in culture media to detect microbial strains capable of producing β-N-acetylglucosaminidase. Colonies that express this enzyme cleave the substrate, resulting in blue staining. This chromogenic response facilitates the identification and isolation of specific bacterial or fungal strains in mixed cultures. The method is advantageous because it provides direct visual confirmation of enzyme expression without requiring further chemical or radioactive labeling.

The substrate also finds utility in biochemical and enzymatic studies, particularly for enzyme activity assays in vitro. Researchers use it to measure the kinetic parameters of β-N-acetylglucosaminidase and to evaluate enzyme inhibition. These studies contribute to the characterization of glycosidases in normal physiology as well as in disease states. Because the chromogenic response is quantifiable, the substrate is suitable for spectrophotometric assays in microplate formats, making it compatible with high-throughput screening systems.

The compound is typically supplied as a lyophilized powder and is stable under standard storage conditions. For use in assays, it is dissolved in aqueous or buffered solutions, and the reaction is usually carried out at slightly acidic to neutral pH, which corresponds to the optimal pH range for β-N-acetylglucosaminidase activity. The oxidation step necessary for color development proceeds under atmospheric oxygen without requiring additional reagents, simplifying the procedure and enhancing reproducibility.

Due to its specificity, ease of use, and clear visual output, 5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-glucosaminide remains an important reagent in diagnostic histochemistry, enzymology, and microbiology. Its ability to reveal enzyme activity at the microscopic level contributes significantly to the understanding of cellular processes involving glycosidase function. The compound is also employed in applied research, including drug development and enzyme engineering, where monitoring of glycosidase activity is essential. Its continued relevance reflects the utility of chromogenic substrates in biochemical research and clinical diagnostics.

References

2000. Distribution of enzyme-bearing cells in GM2 gangliosidosis mice: regionally specific pattern of cellular infiltration following bone marrow transplantation. Acta Neuropathologica, 99(2).
DOI: 10.1007/pl00007420

2011. Applying neutral drift to the directed molecular evolution of a �-glucuronidase into a �-galactosidase: Two different evolutionary pathways lead to the same variant. BMC Research Notes, 4.
DOI: 10.1186/1756-0500-4-138

2020. Genome Mining Reveals the Biosynthetic Pathways of Polyhydroxyalkanoate and Ectoines of the Halophilic Strain Salinivibrio proteolyticus M318 Isolated from Fermented Shrimp Paste. Marine Biotechnology, 22(5).
DOI: 10.1007/s10126-020-09986-z