5-Bromo-4-chloro-3-indolyl phosphate disodium salt
[CAS# 102185-33-1]

Identification

• Classification: Organic raw materials >> Heterocyclic compound >> Indoles
• Name: 5-Bromo-4-chloro-3-indolyl phosphate disodium salt
• Synonyms: disodium (5-bromo-4-chloro-1H-indol-3-yl) phosphate
• Molecular Formula: C₈H₄BrClNO₄P.₂Na
• Molecular Weight: 370.43
• CAS Registry Number: 102185-33-1
• EC Number: 600-286-2
• SMILES: C1=CC(=C(C2=C1NC=C2OP(=O)([O-])[O-])Cl)Br.[Na+].[Na+]

Properties

• Melting point: 300 °C (Expl.)
• Solubility: H2O: 20 mg/mL (Expl.)

Safety Data

• Hazard Symbols: GHS07 Warning
• Risk Statements: H319-H315-H335
• Safety Statements: P261-P271-P280

Discovery and Applications

5-Bromo-4-chloro-3-indolyl phosphate disodium salt is a synthetic chromogenic substrate widely used in molecular biology and biochemistry for the detection of enzymatic activity, particularly that of alkaline phosphatase. This compound is structurally derived from indole, substituted at the 5- and 4-positions with bromine and chlorine atoms, respectively, and features a phosphate ester moiety at the 3-position, forming a disodium salt to enhance water solubility.

The compound was developed as part of efforts to create sensitive and stable substrates for use in immunoassays and blotting techniques. Its utility stems from its ability to yield a colored precipitate upon enzymatic cleavage. In the presence of alkaline phosphatase, 5-bromo-4-chloro-3-indolyl phosphate (BCIP) undergoes dephosphorylation to form an indolyl intermediate, which spontaneously dimerizes and oxidizes in situ to generate a blue-purple precipitate. This visible product enables the qualitative and semi-quantitative detection of phosphatase activity directly on membranes or in tissue samples.

BCIP is frequently used in conjunction with nitro blue tetrazolium (NBT) in colorimetric detection systems. When both substrates are used together, alkaline phosphatase catalyzes the conversion of BCIP, and the oxidized indolyl intermediate reduces NBT to an insoluble dark blue formazan product. This system provides a highly sensitive and permanent signal that is stable under ambient light and storage conditions. It is particularly useful in applications such as Western blotting, in situ hybridization, immunohistochemistry, and enzyme-linked immunosorbent assays (ELISAs), where it allows visualization of antibody or probe binding.

In molecular biology research, BCIP/NBT is often employed as a detection method for probes bound to nucleic acids on nitrocellulose or nylon membranes. The technique facilitates the analysis of gene expression patterns and the presence of specific DNA or RNA sequences. The resulting blue coloration indicates successful hybridization or antigen detection, and its intensity can be correlated to the amount of target molecule present.

In histological studies, BCIP has also been used for localization of phosphatase enzyme activity in tissue samples. The insoluble product formed remains fixed at the site of enzymatic activity, allowing microscopic examination of cellular and tissue-specific expression patterns. This has been valuable in developmental biology, pathology, and the study of cellular differentiation.

The substrate is typically provided as a disodium salt to improve solubility in aqueous buffers and to ensure compatibility with biological samples. It is generally stable under standard storage conditions and can be prepared as a stock solution in water or dimethylformamide for experimental use.

Although highly effective in non-radioactive labeling systems, precautions must be taken when handling BCIP due to its potential as an irritant. Laboratory safety guidelines recommend the use of gloves and protective eyewear, and solutions should be handled in well-ventilated areas. Waste disposal should be conducted in accordance with local environmental regulations due to the generation of halogenated aromatic byproducts.

The development and application of 5-bromo-4-chloro-3-indolyl phosphate disodium salt represent a significant advancement in the field of bioanalytical chemistry, providing researchers with a reliable, sensitive, and visually discernible method to detect and quantify enzyme activity and molecular interactions across a wide range of biological and diagnostic systems.

References

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DOI: 10.1093/emboj/17.9.2619

2014. Discovery of novel enzymes with industrial potential from a cold and alkaline environment by a combination of functional metagenomics and culturing. Microbial Cell Factories, 13(72).
DOI: 10.1186/1475-2859-13-72

2024. Paper Analytical Device for the Colourimetric Detection of Alkaline Phosphatase in Serum and Saliva. Journal of Analysis and Testing, 8(3).
DOI: 10.1007/s41664-024-00304-3