X-α-Gal (5-Bromo-4-chloro-3-indolyl-α-D-galactopyranoside)

X-α-Gal (5-Bromo-4-chloro-3-indolyl-α-D-galactopyranoside, CAS 74783-98-5) is a chromogenic substrate selective for α-galactosidase (EC 3.2.1.22) enzymes, producing an insoluble intense blue precipitate upon hydrolysis that enables clear visualization of enzyme-positive colonies or plaques in microbiological and molecular biology applications. With molecular formula C14H15BrClNO6 (MW 408.63 g/mol), this white crystalline compound features an α-D-galactopyranosyl moiety O-glycosidically linked to the 3-position of 5-bromo-4-chloro-1H-indol-3-ol, remaining colorless until cleaved by α-galactosidases from pathogens like Streptococcus pneumoniae, E. coli, and yeast (Saccharomyces cerevisiae MEL1 gene product), liberating the indoxyl aglycone that spontaneously dimerizes and oxidizes to the highly colored dibromo-dichloro-indigo dye localized to the enzyme source. Primarily utilized in yeast two-hybrid screening as an alternative to X-Gal/β-gal for α-galactosidase reporter systems (e.g., MEL1 promoter), it detects protein-protein interactions by blue colony formation without filter-lift assays, while supporting differential media for α-gal-positive bacteria in clinical diagnostics (pneumococcal detection) and functional screening of GH27/D/Glycosyl hydrolase family enzymes. Its α-anomeric specificity distinguishes it from β-selective X-Gal, providing orthogonal readouts in glycosidase library screening for Fabry disease diagnostics (α-Gal A deficiency), blood group B antigen breakdown, and biofuel research targeting α-galactan hydrolysis.

Appearance

• White to off-white crystalline powder.
• Hydrolysis produces intense blue-green insoluble precipitate.​

Source

• Koenigs-Knorr synthesis using protected α-D-galactopyranosyl halide + bromochloroindol-3-ol.
• Commercial microbiology grade (GoldBio, MedChemExpress >98%).

Molecular Weight and Structure

• Formula: C14H15BrClNO6; MW 408.63 g/mol.
• (2R,3R,4S,5S,6S)-2-(5-bromo-4-chloro-1H-indol-3-yloxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol.​

Sugar Specificity

• Selective α-D-galactopyranoside for α-galactosidases (GH27/36/D).
• Minimal hydrolysis by β-galactosidases.​

Biological Activity

• Chromogenic reporter for MEL1/α-galactosidase activity.
• Non-toxic; detects enzyme via localized blue product.​

Purity and Microbial Contamination

• ≥98% (HPLC); molecular biology grade.
• Synthetic reagent; non-sterile.​

Identity and Quality Control

• [α]D +120° to +130° (c=1, DMF); mp >230°C (dec).
• Characteristic blue-green hydrolysis product.​

Shelf Life and Storage

• 3 years -20°C desiccated; DMF/DMSO stock stable indefinitely.
• Light/moisture protected.​

Applications

• Yeast two-hybrid α-galactosidase screening; pneumococcal detection.
• Fabry disease diagnostics; GH enzyme library screening.

Key Characteristics

• α-Galactose specific; blue-green colonies/plaques.
• No-wash; orthogonal to X-Gal/β-gal systems.​

Citation Links

• https://goldbio.com/product/1972/x-a-gal​
• https://www.goldbio.com/products/x-a-gal​
• https://www.medchemexpress.com/x-alpha-gal.html​
• https://zellx.de/product/5-bromo-4-chloro-3-indolyl-α-d-glucopyranoside-x-α-glc/​
• https://www.sigmaaldrich.com/US/en/product/sigma/73837​
• https://pubchem.ncbi.nlm.nih.gov/compound/2733916​
• https://www.srlchem.com/product/details/25/74021/5-Bromo-4-Chloro-3-Indolyl-a-D-Glucopyranoside​
• https://www.chemicalbook.com/ChemicalProductProperty_EN_CB8358680.htm​
• https://www.sigmaaldrich.com/LT/en/product/sigma/73837​
• https://pubmed.ncbi.nlm.nih.gov/3575911/