5-Bromo-4-chloro-3-indolyl 2-acetamido-2-deoxy-b-D-glucopyranoside

5-Bromo-4-chloro-3-indolyl N-acetyl-beta-D-glucosaminide is a chemical compound that has become widely known for its applications in scientific experiments. It is a reagent that is used in histochemistry of mammal tissues to detect the presence of beta-galactosidase activity. In this paper, we will discuss various aspects related to 5-Bromo-4-chloro-3-indolyl N-acetyl-beta-D-glucosaminide.

Definition and Background:

5-Bromo-4-chloro-3-indolyl N-acetyl-beta-D-glucosaminide is also known as X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). It is a substrate for β-galactosidase enzyme that cleaves its glycosidic linkage and converts X-gal into insoluble blue colored (2-(2-hydroxy-1,3-dihydro-3-oxo-1H-inden-2-ylidene)-2-oxoethyl) 2-acetamido-2-deoxy-β-D-glucopyranoside. The compound was first introduced by Miller in 1972, and since then, it has become a popular substrate for detecting β-galactosidase activity in various molecular biology experiments.

Physical and Chemical Properties:

The molecular formula of 5-Bromo-4-chloro-3-indolyl N-acetyl-beta-D-glucosaminide is C14H15BrClNO6. It is a white to light yellow crystalline powder that is soluble in dimethylformamide, dimethyl sulfoxide, and acetonitrile. The melting point of X-gal is 212-215 °C. The compound has a molecular weight of 408.63 g/mol. Its chemical structure is shown below:

Synthesis and Characterization:

5-Bromo-4-chloro-3-indolyl N-acetyl-beta-D-glucosaminide can be synthesized by several methods. The most common method involves the reaction of 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside with acetic anhydride in the presence of pyridine. The resulting product is then treated with N-acetylglucosamine and purified by recrystallization. The synthesis of X-gal is shown below:

The characterization of 5-Bromo-4-chloro-3-indolyl N-acetyl-beta-D-glucosaminide can be done by various analytical techniques such as NMR spectroscopy and mass spectrometry.

Analytical Methods:

The detection of β-galactosidase activity using X-gal can be done by various analytical methods such as spectrophotometry, fluorometry, and microscopy. In spectrophotometric analysis, the product of β-galactosidase activity, i.e., 2-(2-hydroxy-1,3-dihydro-3-oxo-1H-inden-2-ylidene)-2-oxoethyl) 2-acetamido-2-deoxy-β-D-glucopyranoside, absorbs light at 570 nm, which can be quantified using a spectrophotometer. In fluorometric analysis, the same product of β-galactosidase activity emits fluorescence that can be measured with a fluorometer.

Biological Properties:

5-Bromo-4-chloro-3-indolyl N-acetyl-beta-D-glucosaminide is not toxic to cells, and it does not affect the viability or proliferation of cells. It is widely used as a reagent to detect β-galactosidase activity in various biological systems. X-gal has been used for the detection of β-galactosidase activity in bacterial cells, mammalian cells, and transgenic animals.

Toxicity and Safety in Scientific Experiments:

5-Bromo-4-chloro-3-indolyl N-acetyl-beta-D-glucosaminide is not toxic to cells, and it is generally safe to use in scientific experiments. However, it should be handled with care, and its use should be in accordance with the standard laboratory protocols.

Applications in Scientific Experiments:

5-Bromo-4-chloro-3-indolyl N-acetyl-beta-D-glucosaminide has become a popular substrate for detecting β-galactosidase activity in various molecular biology experiments. It is used to detect gene expression under the control of a promoter that drives the expression of β-galactosidase. X-gal is also used to detect the expression of a recombinant protein that is fused to β-galactosidase.

Current State of Research:

Currently, research is focused on improving the sensitivity and specificity of X-gal-based assays. New substrates are being developed that can detect activity of other enzymes with similar glycosidic linkages. One such example is 5-bromo-4-chloro-3-indoxyl synthetic glucuronic acid (X-GlcA) that is used to detect β-glucuronidase activity.

Potential Implications in Various Fields of Research and Industry:

The use of 5-Bromo-4-chloro-3-indolyl N-acetyl-beta-D-glucosaminide has a wide range of implications in various fields of research and industry. It can be used to study gene regulation, protein expression, and the development of transgenic animals. X-gal-based assays can also be used for the diagnosis of various diseases, such as lysosomal storage disorders.

Limitations and Future Directions:

One of the limitations of X-gal-based assays is that they are not suitable for real-time monitoring of gene expression. Currently, research is focused on developing new reagents that can detect β-galactosidase activity in live cells.

Future directions in the research of 5-Bromo-4-chloro-3-indolyl N-acetyl-beta-D-glucosaminide include the development of new substrates that can detect activity of other enzymes with similar glycosidic linkages. Additionally, the synthesis of X-gal analogs with improved properties such as solubility and stability is an area of active research. Finally, the application of X-gal-based assays in the diagnosis and treatment of diseases is another area of future research.

COA:

Name: 5-Bromo-4-chloro-3-indolyl 2-acetamido-2-deoxy-b-D-glucopyranose 

CAS: 4264-82-8 or 5609-91-6      M.F.: C₁₆H₁₈BrClN₂O₆      M.W._: 449.68  

*Chromogenic substrate for detecting N-acetyl-β-D-glucosaminidase