Blue‑α‑Gal

Blue‑α‑Gal (CAS 1226578‑88‑6), systematically 5‑bromo‑3‑indolyl α‑D‑galactopyranoside, is a synthetic α‑D‑galactosidase‑specific chromogenic substrate in which a 5‑bromo‑3‑indoxyl aglycone is linked via an α‑glycosidic bond to D‑galactose. When hydrolyzed by α‑D‑galactosidase (EC 3.2.1.22) or other α‑galactoside‑cleaving enzymes, the indoxyl moiety is released and undergoes aerial oxidation to form an insoluble blue indigo‑type precipitate, enabling clear visual detection of enzyme activity. This substrate is used in selective agar media to screen and identify α‑galactosidase‑positive microorganisms, in enzyme‑kinetic assays for recombinant or purified α‑D‑galactosidases, and in histochemical staining for α‑galactosidase localization in glycoproteins and glycolipids. The blue color provides good contrast against background pigmentation, especially in dense or mixed‑culture platings, improving sensitivity and ease of readout. Compared with traditional β‑galactosidase substrates, Blue‑α‑Gal is tailored for α‑galactosidase‑specific systems, making it suitable for α‑galactosidase‑related research and diagnostic applications.

Appearance

• Off‑white to pale yellow crystalline or lyophilized powder.
• Fine, free‑flowing texture suitable for accurate weighing and dissolution in aqueous or mixed aqueous‑organic solvents.

Source / synthesis

• Synthetically produced by coupling 5‑bromo‑3‑indoxyl with α‑D‑galactose under glycosylation conditions to form the α‑D‑galactopyranoside derivative.
• Commercially manufactured by specialty enzyme‑substrate and biochemical suppliers (e.g., Shenzhen GeneSeqTools, ChemicalBook‑linked vendors) as a high‑purity, research‑grade reagent.

Molecular weight and structure

• Molecular formula: C14H16BrNO6C14H16BrNO6.
• Molecular weight: 374.18 g/mol.
• Structure: 5‑bromo‑1H‑indol‑3‑yl α‑D‑galactopyranoside, with an α‑glycosidic linkage between the indole C3‑O and the D‑galactose ring in the (2R,3R,4S,5R,6R) configuration.

Sugar specificity

• Specifically hydrolyzed by α‑D‑galactosidases (EC 3.2.1.22) and other α‑galactoside‑cleaving enzymes.
• Shows minimal cross‑reactivity with β‑glycosidases and other α‑glycosidases under optimized assay conditions, providing high selectivity for α‑D‑galactosidases.

Biological activity

• Acts as a chromogenic reporter for α‑D‑galactosidase‑mediated hydrolysis of terminal α‑D‑galactosyl linkages in glycoproteins, glycolipids, and oligosaccharides.
• Enzymatic cleavage yields 5‑bromo‑3‑indoxyl, which oxidizes and dimerizes into an insoluble blue dye, enabling sensitive detection in bacterial colonies, enzyme assays, and histochemical preparations.

Purity and microbial contamination

• Typically supplied at ≥99% purity as determined by HPLC or similar analytical methods, with product data sheets from major suppliers.
• Indicated as low‑contamination, non‑sterile material suitable for microbiological and biochemical use when handled under standard sterile‑practice conditions.

Identity and quality control

• Identity confirmed by molecular formula, molecular weight, NMR, and HPLC profiles consistent with 5‑bromo‑3‑indolyl α‑D‑galactopyranoside.
• Quality‑control procedures include checks for appearance, solubility, spectral characteristics, and performance in a defined α‑galactosidase activity assay.

Shelf life and storage

• Shelf life is generally 12–24 months when stored as a dry powder at –20 °C in a tightly sealed container, protected from moisture and light.
• Stock solutions in DMSO or DMF should be prepared immediately before use or stored frozen for short periods to minimize degradation and nonspecific oxidation of the indoxyl moiety.

Application

• Screening and phenotypic differentiation of α‑D‑galactosidase‑producing microorganisms (e.g., yeasts and bacteria) on selective agar media.
• Enzyme‑activity assays for recombinant or purified α‑D‑galactosidases in solution or immobilized formats.
• Histological or cytochemical localization of α‑D‑galactosidase activity in tissue sections and glycan‑remodeling studies.

Key characteristics

• High sensitivity and low background due to formation of an insoluble blue dye upon α‑D‑galactosidase hydrolysis.
• Defined α‑D‑galactopyranoside linkage providing selectivity for α‑D‑galactosidases over other glycosidases.
• Crystalline, stable powder with a known molecular formula and weight, suitable for quantitative enzyme assays and standardized media formulations.

Citations 

• ChemicalBook – 5‑Bromo‑3‑indolyl‑α‑D‑galactopyranoside (Blue‑α‑Gal, CAS 1226578‑88‑6, ≥99%): https://www.chemicalbook.com/ProdSupplierGNCB8637659.htm
• Shenzhen GeneSeqTools – 5‑Bromo‑3‑indolyl‑α‑D‑galactopyranoside (Blue‑α‑Gal) product listing: http://www.geneseqtools.com/en/product02_cid_215.html
• ChemicalBook – 5‑Bromo‑3‑indolyl‑α‑D‑galactopyranoside entry (CAS 1226578‑88‑6, MF, purity): https://www.chemicalbook.com/ChemicalProductProperty_US_CB1739769.aspx
• Supplier product page (5‑Bromo‑3‑indolyl‑α‑D‑galactopyranoside, ≥99%, bulk packaging): https://m.chemicalbook.com/ShowSupplierProductsList30896/0_EN.htm
• 5‑Bromo‑3‑indolyl‑β‑D‑galactopyranoside (Bluo‑Gal) properties (for dye‑chemistry and handling context): https://www.glpbio.com/5-bromo-3-indolyl-946-d-galactopyranoside.html
• Fisher Scientific – 5‑Bromo‑3‑indolyl‑β‑D‑galactopyranoside product page (for α‑galactosidase‑substrate reference): https://www.fishersci.com/shop/products/5-bromo-3-indolyl-beta-d-galactopyranoside-98-thermo-scientific/AAJ65978MC
• Nature – Functional characterization of α‑Gal producing lactic acid bacteria (background for α‑Gal and α‑galactosidase systems): https://www.nature.com/articles/s41598-022-11632-8
• PMC – Synthesis of C‑linked α‑Gal and α‑GalNAc (for α‑galactosyl‑moiety and glycosidase‑substrate concepts): https://pmc.ncbi.nlm.nih.gov/articles/PMC8356801/
• General glycosidase‑substrate catalog (chromogenic α‑galactosidase‑substrate overview): https://www.medchemexpress.com/ (search for “α‑galactosidase substrate” or “Blue‑α‑Gal”)
• Enzyme‑substrate database (α‑galactosidase‑chromogenic‑substrates, including 5‑bromo‑3‑indolyl‑α‑D‑galactopyranoside): https://www.chemicalbook.com/ (search for “1226578‑88‑6”)