Rouge‑Gal (CAS 207727‑11‑5), systematically 6‑fluoro‑3‑indolyl β‑D‑galactopyranoside, is a synthetic β‑galactosidase‑specific chromogenic substrate in which a 6‑fluoro‑3‑indolyl (indoxyl) aglycone is linked via a β‑glycosidic bond to D‑galactose. Upon hydrolysis by β‑galactosidase (EC 3.2.1.23, LacZ) in bacterial or recombinant systems, the indoxyl moiety is released and oxidizes to form an insoluble red‑magenta precipitate, enabling clear visual detection of enzyme activity. The distinctly red‑magenta hue offers spectral separation from blue‑forming substrates, making Rouge‑Gal useful for multiplex assays, double‑labeling systems, or co‑detection with other chromogenic reporters. The compound is typically used in aqueous or buffer‑containing agar media for blue–white or color‑based selection of recombinant clones, in enzyme‑kinetic assays, and in histochemical detection of β‑galactosidase expression. Rouge‑Gal is compatible with standard molecular‑biology and microbiological workflows and is supplied as a crystalline, high‑purity powder optimized for chromogenic solid‑medium and cell‑based applications.

Appearance

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

Source / synthesis

• Synthetically produced by coupling 6‑fluoro‑3‑indoxyl with β‑D‑galactose under glycosylation conditions to form the β‑D‑galactopyranoside derivative.
• Commercially manufactured by specialty enzyme‑substrate and carbohydrate‑chemistry suppliers (e.g., Glycosynth, Shenzhen GeneSeqTools) as a high‑purity, research‑grade reagent.

Molecular weight and structure

• Molecular formula: C14H16FNO6C14​H16​FNO6​.
• Molecular weight: 313.28 g/mol.
• Structure: 6‑fluoro‑1H‑indol‑3‑yl β‑D‑galactopyranoside, with a β‑glycosidic linkage between the indole C3‑O and the D‑galactose ring.

Sugar specificity

• Specifically hydrolyzed by β‑galactosidases (LacZ, EC 3.2.1.23) and other β‑galactoside‑cleaving enzymes.
• Shows minimal cross‑reactivity with α‑glycosidases and many other glycosidases under standard cloning‑medium conditions, making it suitable for selective detection of β‑galactosidase activity.

Biological activity

• Acts as a chromogenic reporter for β‑galactosidase‑mediated hydrolysis of β‑D‑galactosyl linkages in lactose analogs and glycosides.
• Enzymatic cleavage yields 6‑fluoro‑3‑indoxyl, which oxidizes and dimerizes into a red‑magenta‑colored, water‑insoluble dye, enabling sensitive detection in bacterial colonies, enzyme assays, and histochemical preparations.

Purity and microbial contamination

• Typically supplied at ≥95–99% purity as indicated by supplier data sheets (e.g., HPLC‑assayed “high‑purity raw material for scientific research”).
• Described as non‑sterile, research‑grade material with low microbial contamination status, suitable for use in standard microbiological and biochemical workflows when handled under aseptic conditions.

Identity and quality control

• Identity inferred from molecular formula, molecular weight, and spectral data (UV/Vis, NMR, and HPLC) consistent with 6‑fluoro‑3‑indolyl β‑D‑galactopyranoside (Rouge‑Gal).
• Quality‑control procedures include checks for appearance, solubility, chromatographic purity, and performance in a defined β‑galactosidase (LacZ) activity assay on agar plates.

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 DMF, DMSO, or methanol should be prepared immediately before use or stored frozen for short periods to minimize degradation and nonspecific oxidation of the indoxyl moiety.

Application

• Color‑based or multiplex selection of recombinant bacterial clones (lacZ⁺ vs lacZ⁻) on agar plates where red‑magenta staining is preferred over blue dyes.
• Chromogenic β‑galactosidase assays in solution or on solid media for enzyme‑kinetic studies and activity profiling.
• Use in histochemical and cell‑based reporter‑gene assays where red‑magenta coloration improves contrast and co‑detection with other chromogens.

Key characteristics

• Produces a red‑magenta insoluble dye upon β‑galactosidase hydrolysis, offering a spectrally distinct alternative to traditional blue‑forming substrates.
• Defined β‑D‑galactopyranoside linkage with a 6‑fluoro‑indolyl aglycone, providing high selectivity for β‑galactosidases over many other glycosidases.
• Crystalline, stable powder with a known molecular formula and weight, suitable for quantitative cloning, enzyme‑assay, and multiplex‑detection applications.

Citations

• Glycosynth – Safety Data Sheet for 6‑Fluoro‑3‑indolyl‑β‑D‑galactopyranoside (Rouge™‑gal, CAS 207727‑11‑5): https://www.glycosynth.co.uk/userdata/files/86045-SDS.pdf
• Shenzhen GeneSeqTools – Rouge‑Gal listing (CAS 207727‑11‑5, 6‑fluoro‑3‑indolyl‑β‑D‑galactopyranoside): http://geneseqtools.com/cn/product03_cid_215_pid_540.html
• GeneSeqTools product overview (Rouge‑Gal alongside other chromogenic β‑galactosidase substrates): http://www.geneseqtools.com/en/product02_cid_215.html
• 5‑Bromo‑3‑indolyl‑β‑D‑galactopyranoside (Bluo‑Gal) product page (for β‑galactosidase‑substrate context and assay principles): https://www.glpbio.com/5-bromo-3-indolyl-946-d-galactopyranoside.html
• Sigma‑Aldrich – 5‑Bromo‑3‑indolyl‑β‑D‑galactopyranoside (X‑Gal‑class substrate, for dye‑chemistry reference): https://www.sigmaaldrich.com/US/en/product/sigma/b4387
• Biotium – Red‑β‑D‑Gal (5‑bromo‑6‑chloro‑3‑indolyl‑β‑D‑galactopyranoside) chromogenic substrate overview (for red‑magenta‑dye background): https://biotium.com/product/red-b-d-gal-5-bromo-6-chloro-3-indolyl-b-d-galactopyranoside/
• ScienceDirect – X‑Gal overview (for general chromogenic β‑galactosidase‑substrate context): https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/x-gal
• abcam – Magenta‑Gal chromogenic β‑galactosidase substrate (for magenta‑dye and cloning‑application context): https://www.abcam.com/en-us/products/biochemicals/magenta-gal-chromogenic-beta-galactosidase-substrate-ab146381
• General glycosidase‑substrate catalog (Rouge‑Gal and related indolyl‑β‑D‑galactosides overview): https://www.chemicalbook.com/ (search for “207727‑11‑5”)
• Red‑β‑D‑Gal and related indolyl‑β‑galactosides product page (for dye‑color and assay‑format reference): https://biotium.com/product/red-b-d-gal-5-bromo-6-chloro-3-indolyl-b-d-galactopyranoside/