5-Bromo-6-chloro-3-indolyl b-D-glucopyranoside

Magenta b-D-Glc; Magenta b-D-glucoside

5-Bromo-6-chloro-3-indolyl caprylate, also known as X-gal, is a chemical compound that is commonly used as a histochemical substrate for the detection of β-galactosidase (β-gal) activity. It is an off-white to light brown powder that is soluble in dimethyl sulfoxide, dimethylformamide, and N,N-dimethylacetamide. The chemical formula of X-gal is C14H15BrClNO6, and its molecular weight is 408.63 g/mol. In this paper, we will discuss the physical and chemical properties of X-gal, its synthesis and characterization, analytical methods used for its detection, its biological properties, toxicity, and safety in scientific experiments, its applications in scientific experiments, the current state of research, its potential implications in various fields of research and industry, limitations, and future directions.

Physical and Chemical Properties

5-Bromo-6-chloro-3-indolyl caprylate is a crystalline powder that has a melting point of 221-227°C. It is slightly soluble in water, ethanol, and methanol but is soluble in most organic solvents. The compound is stable when stored in a dry, cool, and dark place. It is non-carcinogenic and non-toxic to most organisms.

Synthesis and Characterization

5-Bromo-6-chloro-3-indolyl caprylate can be synthesized by reacting 5-bromo-6-chloroindole with the protected galactose derivative, 1-O-acetyl-2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl bromide, in the presence of silver oxide in dichloromethane. The reaction yields two products, 5-Bromo-6-chloro-3-indolyl caprylate and a side product, 6-chloro-5-hydroxyindole.

The characterization of 5-Bromo-6-chloro-3-indolyl caprylate can be done using various techniques such as NMR spectroscopy, mass spectrometry, infrared spectroscopy, and X-ray crystallography. NMR spectroscopy can be used to determine the purity of the compound, the structure of the molecule, and the position of the substituents in the indole ring. Mass spectrometry can be used to determine the molecular weight of the compound, while infrared spectroscopy can be used to determine the functional groups present in the molecule.

Analytical Methods

5-Bromo-6-chloro-3-indolyl caprylate is commonly used as a substrate for β-gal detection in molecular biology research. It is used to screen for the presence of recombinant plasmids in bacterial colonies or to detect gene expression in mammalian cells. The detection of 5-Bromo-6-chloro-3-indolyl caprylate hydrolysis by β-gal leads to the formation of a blue product.

Several analytical methods are used for the detection of 5-Bromo-6-chloro-3-indolyl caprylate hydrolysis, including spectrophotometry, fluorescence, and chromogenic assays. Spectrophotometric assays are based on the colorimetric detection of the blue product formed in the hydrolysis reaction. Fluorescence assays are based on the detection of the fluorescence emitted by the blue product under UV light. Chromogenic assays are based on the detection of the yellow product formed by the hydrolysis of 5-Bromo-6-chloro-3-indolyl caprylate in the presence of ferric ions.

Biological Properties

5-Bromo-6-chloro-3-indolyl caprylate is widely used in biological research as a substrate for the detection of β-gal activity. β-gal is an enzyme that catalyzes the hydrolysis of lactose into glucose and galactose. In molecular biology, β-gal is used as a reporter gene to monitor the expression of target genes. The expression of the target gene is monitored by the detection of β-gal activity using 5-Bromo-6-chloro-3-indolyl caprylate as a substrate.

Toxicity and Safety in Scientific Experiments

5-Bromo-6-chloro-3-indolyl caprylate is generally considered safe for use in scientific experiments. It is not mutagenic, teratogenic, or carcinogenic. However, it should be handled with care and should not be ingested or inhaled. The material safety data sheet (MSDS) for 5-Bromo-6-chloro-3-indolyl caprylate should be consulted before handling the compound.

Applications in Scientific Experiments

5-Bromo-6-chloro-3-indolyl caprylate is widely used in molecular biology research as a substrate for the detection of β-gal activity. It is used to screen for the presence of recombinant plasmids in bacterial colonies or to detect gene expression in mammalian cells. 5-Bromo-6-chloro-3-indolyl caprylate can also be used to visualize and quantify gene expression patterns in developing embryos.

Current State of Research

5-Bromo-6-chloro-3-indolyl caprylate has been extensively used in molecular biology research for over 40 years. However, there is ongoing research aimed at improving the detection sensitivity and specificity of 5-Bromo-6-chloro-3-indolyl caprylate-based assays. New β-gal substrates with improved properties are being developed, including substrates that are resistant to endogenous β-gal activity, substrates that have improved sensitivity, and substrates that can be used in living cells.

Potential Implications in Various Fields of Research and Industry

5-Bromo-6-chloro-3-indolyl caprylate has potential implications in various fields of research and industry, including gene therapy, drug development, and biotechnology. In gene therapy, 5-Bromo-6-chloro-3-indolyl caprylate can be used as a reporter gene to monitor gene expression in vivo. In drug development, 5-Bromo-6-chloro-3-indolyl caprylate-based assays can be used to screen compounds for their effect on gene expression. 5-Bromo-6-chloro-3-indolyl caprylate can also be used in biotechnology for the production of recombinant proteins.

Limitations and Future Directions

One of the limitations of 5-Bromo-6-chloro-3-indolyl caprylate is that it can only detect β-gal activity in fixed cells, tissues, or bacterial colonies. 5-Bromo-6-chloro-3-indolyl caprylate cannot be used to monitor β-gal activity in vivo. Furthermore, 5-Bromo-6-chloro-3-indolyl caprylate-based assays can produce false positives due to endogenous β-gal activity. Therefore, the development of improved substrates that are specific for recombinant β-gal is a direction for future research. Other future directions include the development of substrates that are compatible with live-cell imaging and the development of substrates that can be used in high-throughput screening assays.

Conclusion

In conclusion, 5-Bromo-6-chloro-3-indolyl caprylate is a widely used chemical compound in molecular biology research that has many applications in scientific experiments. It is an effective substrate for the detection of β-gal activity and has potential implications in various fields of research and industry. While there are limitations to its use, ongoing research is focused on improving the specificity and sensitivity of 5-Bromo-6-chloro-3-indolyl caprylate-based assays and developing new substrates for the detection of β-gal activity.