5-Bromo-4-chloro-3-indolyl-beta-D-glucuronide cyclohexylammonium salt
[CAS# 114162-64-0]

Identification

• Classification: Biochemical >> Carbohydrate >> Monosaccharide
• Name: 5-Bromo-4-chloro-3-indolyl-beta-D-glucuronide cyclohexylammonium salt
• Synonyms: X-Gluc; (2S,3S,4S,5R,6S)-6-[(5-bromo-4-chloro-1H-indol-3-yl)oxy]-3,4,5-trihydroxyoxane-2-carboxylic acid;cyclohexanamine
• Molecular Formula: C₁₄H₁₃BrClNO₇.C₆H₁₃N
• Molecular Weight: 521.79
• CAS Registry Number: 114162-64-0
• EC Number: 601-302-0
• SMILES: C1CCC(CC1)N.C1=CC(=C(C2=C1NC=C2O[C@H]3[C@@H]([C@H]([C@@H]([C@H](O3)C(=O)O)O)O)O)Cl)Br

Properties

• Melting point: 230 °C (Expl.)

Safety Data

• Hazard Symbols: GHS07 Warning
• Risk Statements: H302-H312-H332
• Safety Statements: P261-P264-P270-P271-P280-P301+P317-P302+P352-P304+P340-P317-P321-P330-P362+P364-P501

Discovery and Applications

5-Bromo-4-chloro-3-indolyl-beta-D-glucuronide cyclohexylammonium salt is a synthetic chemical compound widely used as a chromogenic substrate in enzymatic assays, particularly for detecting and measuring the activity of β-glucuronidase enzymes. This compound belongs to the class of indoxyl glucuronides, which release colored products upon enzymatic hydrolysis, facilitating visual or spectrophotometric detection.

The compound consists of a 5-bromo-4-chloro-3-indolyl moiety linked via a glycosidic bond to β-D-glucuronic acid. The cyclohexylammonium component serves as a counterion to improve the compound’s solubility and stability in aqueous solutions, enhancing its usability in biological assays.

The discovery and development of this substrate are rooted in efforts to create sensitive and specific indicators for β-glucuronidase, an enzyme involved in the hydrolysis of glucuronides. β-Glucuronidase is present in a variety of biological systems, including bacteria, mammals, and plants, and plays a role in the metabolism of complex carbohydrates and detoxification processes. In microbiology and clinical diagnostics, β-glucuronidase activity serves as a marker for certain bacterial species, including Escherichia coli, making substrates like 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide valuable tools for detecting microbial contamination.

When β-glucuronidase acts on this substrate, it cleaves the glycosidic bond, releasing 5-bromo-4-chloro-3-indoxyl, which spontaneously oxidizes and dimerizes to form an insoluble blue dye, commonly referred to as indigo dye. This color change provides a clear and easily interpretable signal indicating enzyme presence and activity. This feature makes the compound useful in histochemical staining, microbial detection on culture media, and enzyme activity assays.

Applications of 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide cyclohexylammonium salt span several scientific and industrial fields. In microbiology, it is incorporated into agar media to facilitate the rapid identification of β-glucuronidase-positive bacteria. This allows for the differentiation of Escherichia coli from other coliform bacteria based on the characteristic blue coloration formed by enzyme activity. The substrate’s sensitivity and specificity improve the accuracy and speed of microbial diagnostics in clinical and environmental samples.

In molecular biology and biochemistry, the substrate is utilized to monitor β-glucuronidase reporter gene expression in various experimental systems. The enzymatic cleavage of the substrate serves as a readout for promoter activity, gene expression regulation, and cellular localization studies. The chromogenic nature of the reaction allows for both qualitative and quantitative assessment, aiding in gene function analysis and screening applications.

In addition to its role in enzyme assays, this compound has been used in histochemistry to visualize β-glucuronidase activity in tissue sections. The resulting blue precipitate highlights enzyme distribution, assisting in studies of tissue differentiation, disease states, and developmental biology. Its application in these contexts provides valuable insights into physiological and pathological processes involving glucuronidase enzymes.

The use of 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide cyclohexylammonium salt requires proper handling to maintain substrate integrity and ensure reliable results. It is typically stored in dry, cool conditions and dissolved immediately prior to use. Its stability in aqueous media depends on pH and temperature, factors that must be optimized during experimental procedures.

Overall, 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide cyclohexylammonium salt is a well-established biochemical reagent with significant utility in enzymology, microbiology, molecular biology, and histology. Its specific interaction with β-glucuronidase and the resultant chromogenic response make it an essential tool for enzyme detection and activity analysis across various scientific disciplines.

References

1995. Evaluation of new medium with chromogenic substrates for members of the family Enterobacteriaceae in urine samples. Journal of Clinical Microbiology, 33(1).
DOI: 10.1128/jcm.33.1.199-201.1995

1998. Membrane filter method based on FC-5-bromo-4-chloro-3-indolyl-beta-D-glucuronide medium facilitates enumeration of Escherichia coli in foods and poultry carcass rinses. Journal of Food Protection, 61(3).
DOI: 10.4315/0362-028x-61.3.360

2018. Rapid Detection of Escherichia coli in Water Using Sample Concentration and Optimized Enzymatic Hydrolysis of Chromogenic Substrates. Current Microbiology, 75(6).
DOI: 10.1007/s00284-018-1454-8