5-Bromo-4-chloro-3-indolyl-beta-D-galactoside
[CAS# 7240-90-6]

Identification

• Classification: Pharmaceutical intermediate >> Heterocyclic compound intermediate >> Pyran compound
• Name: 5-Bromo-4-chloro-3-indolyl-beta-D-galactoside
• Synonyms: X-Gal
• Molecular Formula: C₁₄H₁₅BrClNO₆
• Molecular Weight: 408.63
• CAS Registry Number: 7240-90-6
• EC Number: 230-640-8
• SMILES: C1=CC(=C(C2=C1NC=C2O[C@H]3[C@@H]([C@H]([C@H]([C@H](O3)CO)O)O)O)Cl)Br

Properties

• Density: 1.9±0.1 g/cm³ Calc.^*
• Melting point: 236 °C (Decomposes) (Expl.)
• Boiling point: 673.9±55.0 °C 760 mmHg (Calc.)^*
• Flash point: 361.3±31.5 °C (Calc.)^*
• Solubility: DMSO: 39ng/mL (Expl.)
• Index of refraction: 1.732 (Calc.)^*
• alpha: -64 ° (c=1, H2O/DMF (1/1))

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

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

Hazard Classification

Hazard	Class	Category Code	Hazard Statement
Acute toxicity	Acute Tox.	4	H332
Acute toxicity	Acute Tox.	4	H312
Acute toxicity	Acute Tox.	4	H302

Discovery and Applications

5-Bromo-4-chloro-3-indolyl-beta-D-galactoside is a synthetic chromogenic substrate widely used in molecular biology and biochemistry to detect the activity of the enzyme β-galactosidase. This compound consists of a substituted indole moiety linked to the sugar beta-D-galactose. The substitutions at the 5 and 4 positions of the indole ring with bromine and chlorine atoms respectively contribute to the distinct colorimetric properties of the molecule.

The discovery of this substrate is rooted in the need for sensitive and specific detection methods for β-galactosidase, an enzyme frequently used as a reporter in gene expression studies. The compound was developed to improve upon earlier substrates by providing clearer and more intense color changes upon enzymatic cleavage.

In enzymatic assays, 5-bromo-4-chloro-3-indolyl-beta-D-galactoside serves as a non-fluorescent molecule that, when hydrolyzed by β-galactosidase, releases a chromophore known as 5-bromo-4-chloro-3-indoxyl. This intermediate undergoes oxidation and dimerization to form an insoluble blue precipitate. The formation of this colored product allows for direct visualization of β-galactosidase activity on agar plates, membranes, or in solution.

This substrate is extensively used in molecular cloning and microbiology for blue-white screening, where recombinant bacterial colonies expressing the lacZ gene can be distinguished from non-recombinant colonies by color. The blue color indicates functional β-galactosidase expression and successful insertion of target DNA sequences into vectors containing the lacZ reporter gene.

In addition to blue-white screening, 5-bromo-4-chloro-3-indolyl-beta-D-galactoside is employed in histochemical staining to localize β-galactosidase activity in tissues or cells. This facilitates studies of gene expression patterns, cellular differentiation, and developmental biology.

The chemical stability and chromogenic properties of the compound, enhanced by the halogen substitutions, make it a preferred choice for sensitive and straightforward detection of β-galactosidase activity. Its insoluble blue product is stable under assay conditions and easily visualized without the need for additional reagents or complex equipment.

In summary, 5-bromo-4-chloro-3-indolyl-beta-D-galactoside is a halogenated chromogenic substrate designed for detecting β-galactosidase activity. It is widely used in molecular biology for blue-white screening and histochemical applications, providing a reliable and visually distinct indication of enzyme presence and activity.

References

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2024. Efficient transformation and genome editing in a nondomesticated, biocontrol strain, Bacillus subtilis GLB191. Phytopathology Research, 6(1).
DOI: 10.1186/s42483-024-00287-0