5-Bromo-6-chloro-3-indolyl-beta-D-galactoside
[CAS# 93863-88-8]

Identification

• Classification: Biochemical >> Carbohydrate >> Monosaccharide
• Name: 5-Bromo-6-chloro-3-indolyl-beta-D-galactoside
• Synonyms: (2S,3R,4S,5R,6R)-2-[(5-bromo-6-chloro-1H-indol-3-yl)oxy]-6-(hydroxymethyl)oxane-3,4,5-triol
• Molecular Formula: C₁₃H₁₅BrClNO₆
• Molecular Weight: 408.63
• CAS Registry Number: 93863-88-8
• EC Number: 679-549-9
• SMILES: C1=C2C(=CC(=C1Br)Cl)NC=C2O[C@H]3[C@@H]([C@H]([C@H]([C@H](O3)CO)O)O)O

Properties

• Density: 1.9±0.1 g/cm³ Calc.^*
• Boiling point: 673.9±55.0 ºC 760 mmHg (Calc.)^*
• Flash point: 361.3±31.5 ºC (Calc.)^*
• Index of refraction: 1.732 (Calc.)^*

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

Safety Data

• Hazard Symbols: GHS07;GHS08 Danger
• Hazard Statements: H315-H319-H335-H370
• Precautionary Statements: P260-P261-P264-P264+P265-P270-P271-P280-P302+P352-P304+P340-P305+P351+P338-P308+P316-P319-P321-P332+P317-P337+P317-P362+P364-P403+P233-P405-P501

Hazard Classification

Hazard	Class	Category Code	Hazard Statement
Eye irritation	Eye Irrit.	2	H319
Specific target organ toxicity - single exposure	STOT SE	1	H370
Skin irritation	Skin Irrit.	2	H315
Specific target organ toxicity - single exposure	STOT SE	3	H335

Discovory and Applicatios

5-Bromo-6-chloro-3-indolyl-β-D-galactoside, commonly abbreviated as X-Gal, is a synthetic organic compound extensively used in molecular biology as a chromogenic substrate for the enzyme β-galactosidase. It is a derivative of galactose in which the aglycone is a substituted indole moiety bearing a bromo and chloro substituent at the 5- and 6-positions, respectively. This chemical is not naturally occurring and was developed as part of advancements in enzyme substrate chemistry for biochemical assays and recombinant DNA technologies.

The primary historical significance of 5-bromo-6-chloro-3-indolyl-β-D-galactoside lies in its utility in the development of blue-white screening, a method introduced in the 1980s for detecting recombinant bacterial colonies. In this assay, X-Gal serves as a substrate for β-galactosidase, an enzyme encoded by the lacZ gene. When cleaved by the enzyme, X-Gal undergoes hydrolysis to release 5-bromo-6-chloro-3-hydroxyindole, which spontaneously dimerizes and oxidizes to produce an intensely colored blue indigo dye. This colorimetric change enables researchers to distinguish colonies expressing functional β-galactosidase (blue) from those with a disrupted lacZ gene due to successful DNA insertion (white), providing a powerful tool for cloning and genetic manipulation.

The synthesis of X-Gal involves glycosylation of the substituted indoxyl moiety with a protected galactose derivative, followed by deprotection steps to yield the final product. The critical synthetic challenge is maintaining the stability of the indole system while achieving the desired stereospecificity of the glycosidic bond. The compound is generally supplied as a white to off-white powder, which is soluble in organic solvents like dimethylformamide (DMF) or dimethyl sulfoxide (DMSO), and used in aqueous assay solutions.

In microbiology and genetic engineering, X-Gal is most famously used in conjunction with isopropyl β-D-1-thiogalactopyranoside (IPTG), which acts as an inducer of the lac operon in E. coli. This combination permits the controlled expression of β-galactosidase in bacteria harboring plasmids with lacZ or lacZα sequences. The screening technique has been foundational in recombinant plasmid selection, contributing to developments in gene cloning, synthetic biology, and genome editing technologies.

Apart from its use in bacterial systems, 5-bromo-6-chloro-3-indolyl-β-D-galactoside is employed in histochemical and cytochemical staining protocols to detect β-galactosidase activity in eukaryotic cells and tissues. For example, in gene expression studies involving transgenic animals or gene therapy vectors, X-Gal can be used to visualize reporter gene activity by staining tissue sections, thus mapping gene expression spatially and temporally. This technique has helped elucidate gene function, promoter activity, and lineage tracing in developmental biology.

The stability and performance of X-Gal in biological assays are influenced by environmental conditions such as pH, temperature, and oxygen availability. Under optimal assay conditions, typically near neutral pH and at physiological temperatures, X-Gal yields high contrast results. However, it must be protected from light and air during storage to prevent premature oxidation of the indoxyl intermediate.

From a toxicological perspective, 5-bromo-6-chloro-3-indolyl-β-D-galactoside is considered to have low acute toxicity and is generally handled safely in laboratory settings using standard precautions. Its degradation products, including halogenated indigo dyes, are chemically stable but can accumulate in biological systems if not properly disposed of. Environmental considerations emphasize careful waste management, especially when large-scale assays or screenings are performed.

As a specialized reagent, X-Gal remains indispensable in modern biosciences, particularly in molecular cloning and transgenic research. Its ease of use, clear visual output, and compatibility with a wide range of organisms and systems have ensured its enduring role in laboratories around the world.

References

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2023. Competition in the Phaseolus vulgaris-Rhizobium symbiosis and the role of resident soil rhizobia in determining the outcomes of inoculation. Plant and Soil, 486(1-2).
DOI: 10.1007/s11104-023-05903-0