5-Bromo-6-chloro-3-indolyl b-D-glucuronide cyclohexylammonium salt

5-溴-6-氯-3-吲哚-beta-D-葡萄糖醛酸苷环己胺盐

5-Bromo-6-chloro-3-indolyl b-D-glucuronide cyclohexylammonium salt is a magenta enzyme substrate commonly used in biochemical assays. This compound is a derivative of indole, containing bromine and chlorine atoms at positions 5 and 6, respectively. The glucuronide moiety is attached to the indolyl ring, and the cyclohexylammonium cation serves as a counterion to balance the charge. This substrate is often utilized in the detection and quantification of beta-glucuronidase activity, an enzyme involved in the metabolism of glucuronides. Upon cleavage by beta-glucuronidase, the substrate produces a magenta-colored product, allowing for easy visualization and measurement of enzyme activity.

5-Bromo-6-chloro-3-indolyl-D-glucuronide cyclohexylammonium salt, also known as X-Gluc, is a widely used substrate for the detection of the enzymatic activity of beta-glucuronidase in various scientific experiments. This paper aims to provide a comprehensive overview of X-Gluc, including its definition, physical and chemical properties, synthesis, characterization, analytical methods, biological properties, toxicity and safety in scientific experiments, applications in scientific experiments, current state of research, potential implications in various fields of research and industry, limitations, and future directions.

Definition and Background:

5-Bromo-6-chloro-3-indolyl-D-glucuronide cyclohexylammonium salt is a chromogenic substrate that is widely used to detect the enzymatic activity of beta-glucuronidase (GUS) in various scientific experiments. GUS is a common marker enzyme that is widely used to study gene expression in plants, animals, and bacteria. X-Gluc is preferred over other substrates due to its high sensitivity, stability, and selectivity. X-Gluc is commercially available in different forms, such as powder, tablet, and solution.

Physical and Chemical Properties:

5-Bromo-6-chloro-3-indolyl-D-glucuronide cyclohexylammonium salt has a molecular formula of C20H25BrClN2O9, a molecular weight of 547.8 g/mol, and a melting point of 220-222°C. X-Gluc is a white to off-white solid that is poorly soluble in water, methanol, ethanol, and acetone but soluble in dimethyl sulfoxide (DMSO) and dimethylformamide (DMF). X-Gluc has a maximum absorption at 587 nm and a molar extinction coefficient of 7,400 M-1cm-1.

Synthesis and Characterization:

5-Bromo-6-chloro-3-indolyl-D-glucuronide cyclohexylammonium salt can be synthesized by various methods, including the reaction of 5-bromo-6-chloro-3-indolyl-beta-D-glucuronide with cyclohexylamine hydrochloride in the presence of triethylamine and acetonitrile. The synthesized X-Gluc can be characterized by various techniques, such as nuclear magnetic resonance (NMR), infrared (IR) spectroscopy, and high-performance liquid chromatography (HPLC).

Analytical Methods:

5-Bromo-6-chloro-3-indolyl-D-glucuronide cyclohexylammonium salt can be detected by various analytical methods, including UV-Vis spectroscopy, HPLC, and paper chromatography. UV-Vis spectroscopy is the most commonly used method for detecting X-Gluc due to its high sensitivity and specificity.

Biological Properties:

5-Bromo-6-chloro-3-indolyl-D-glucuronide cyclohexylammonium salt is not toxic to cells and does not interfere with the growth of microorganisms or the viability of cells. X-Gluc is preferentially hydrolyzed by beta-glucuronidase, an enzyme that is widely expressed in various tissues and cells, especially in the liver and kidney. The hydrolysis of X-Gluc by beta-glucuronidase results in the formation of a blue precipitate that can be easily detected by visual inspection or microscopy.

Toxicity and Safety in Scientific Experiments:

5-Bromo-6-chloro-3-indolyl-D-glucuronide cyclohexylammonium salt is generally considered safe for use in scientific experiments. However, like any other chemicals, X-Gluc should be handled with care and under proper laboratory conditions to avoid skin and eye irritation, inhalation, or ingestion. X-Gluc may be harmful if swallowed or absorbed through the skin and may cause respiratory and gastrointestinal irritation or allergic reactions.

Applications in Scientific Experiments:

5-Bromo-6-chloro-3-indolyl-D-glucuronide cyclohexylammonium salt has various applications in scientific experiments, such as in the detection and quantification of beta-glucuronidase activity in various tissues and cells, in the visualization of gene expression patterns in transgenic plants and animals, and in the analysis of bacterial transformation and recombination. X-Gluc is also used in various quality control assays for the detection of bacterial contamination in food and water samples.

Current State of Research:

5-Bromo-6-chloro-3-indolyl-D-glucuronide cyclohexylammonium salt has been extensively studied in various scientific fields, including molecular biology, biochemistry, pharmacology, and toxicology. X-Gluc has been used to study the expression and regulation of various genes in different organisms, such as Arabidopsis thaliana, Escherichia coli, and Streptomyces coelicolor. X-Gluc has also been used in drug discovery and development to screen for potential inhibitors of beta-glucuronidase activity.

Potential Implications in Various Fields of Research and Industry:

5-Bromo-6-chloro-3-indolyl-D-glucuronide cyclohexylammonium salt has potential implications in various fields of research and industry, such as in the development of new diagnostic and therapeutic tools for cancer, genetic disorders, and infectious diseases. X-Gluc can also be used in the detection and quantification of beta-glucuronidase activity in environmental samples, such as soil and water, as well as in industrial processes, such as pharmaceutical and biotechnological production.

Limitations and Future Directions:

5-Bromo-6-chloro-3-indolyl-D-glucuronide cyclohexylammonium salt has some limitations, such as its poor solubility, limited stability, and background interference in some samples. Future research directions include the development of new X-Gluc analogs that exhibit higher solubility, stability, and selectivity, the optimization of the reaction conditions for detecting beta-glucuronidase activity, and the exploration of new applications of X-Gluc in various fields of research and industry.

Future Directions:

1. Exploring X-Gluc's potential as a substrate for detecting other enzymes

2. Developing X-Gluc analogs with improved solubility and stability

3. Investigating X-Gluc's potential for detecting beta-glucuronidase activity in complex biological samples

4. Optimizing the reaction conditions for detecting beta-glucuronidase activity using X-Gluc

5. Developing X-Gluc-based assays for high-throughput screening of beta-glucuronidase inhibitors

6. Investigating X-Gluc's potential role in studying the regulation of gene expression in different organisms

7. Developing X-Gluc-based biosensors for detecting environmental toxins and pollutants

8. Studying the pharmacokinetics and pharmacodynamics of X-Gluc in vivo

9. Investigating the potential of X-Gluc for detecting beta-glucuronidase activity in cancer cells and tissues

10. Developing X-Gluc-based diagnostic tools for genetic disorders and infectious diseases.