FMGal

FMGal, systematically fluorescein‑mono‑β‑D‑galactoside (CAS 102286‑67‑9), is a synthetic β‑galactosidase‑specific fluorogenic substrate in which a fluorescein aglycone is linked via a β‑glycosidic bond to the anomeric carbon of β‑D‑galactopyranose. When hydrolyzed by β‑galactosidase (EC 3.2.1.23) or other β‑galactoside‑cleaving enzymes, the glycosidic bond is cleaved to release fluorescein and β‑D‑galactose. The released fluorescein exhibits strong green fluorescence under visible‑light excitation, allowing highly sensitive, continuous monitoring of enzyme activity in microplate‑based assays. FMGal is widely used in enzymology to characterize β‑galactosidase activity in bacterial, fungal, plant, and mammalian systems, to study enzyme‑substrate specificity among galactosyl‑glycosides, and to quantify activity in kinetic experiments (Km, Vmax) and inhibitor‑screening assays. The compound is supplied as a high‑purity, water‑or slightly DMSO‑soluble powder and is suitable for reporter‑gene systems (e.g., lacZ or β‑galactosidase‑complementation assays), in situ cell‑based imaging, and diagnostic‑type β‑galactosidase‑activity measurements. Its high quantum yield and compatibility with standard fluorometers and microplate readers make FMGal a preferred tool for sensitive, fluorimetric detection of β‑galactosidase activity.

Appearance

• Off‑white to pale yellow crystalline or amorphous solid.
• Forms a clear, colorless to slightly yellow solution in aqueous or buffer‑containing systems; develops bright green fluorescence only after enzymatic hydrolysis.

Source / synthesis

• Synthetically prepared by glycosylation of fluorescein with β‑D‑galactopyranose or a protected β‑D‑galactoside, followed by deprotection and crystallization from water or mixed solvents.
• Commercially supplied by biochemical‑reagent and fluorophore‑chemistry vendors (e.g., Fluorochem, Glentham Life Sciences, various Chinese‑based suppliers) as a research‑grade β‑galactosidase substrate.

Molecular weight and structure

• Molecular formula: C26H22O10C26H22O10.
• Molecular weight: 494.45 g/mol.
• Structure: fluorescein‑mono‑β‑D‑galactoside, with a β‑glycosidic linkage between the aromatic fluorescein group and the anomeric oxygen of the D‑galactose ring in the pyranose form.

Sugar specificity

• Specifically hydrolyzed by β‑galactosidases and β‑galactoside‑cleaving glycosidases, with activity commonly measured at neutral pH.
• Widely used in fluorogenic panels to distinguish β‑galactosidase activity from other glycosidases in microbial and mammalian systems.

Biological activity

• Serves as a fluorogenic reporter whose rate of fluorescein release under defined pH and temperature conditions reflects β‑galactosidase activity.
• Enables determination of kinetic parameters (Km, Vmax), inhibitor‑IC₅₀, and comparative activity profiling of different β‑galactosidases, including those used in lacZ reporter‑gene and in situ assays.

Purity and microbial contamination

• Typically supplied at ≥97–99% purity (e.g., “high purity”, “≥99% HPLC”) as indicated by supplier data sheets and product‑specification labels.
• Described as a non‑sterile, research‑grade solid suitable for use in standard enzymatic assays under clean‑laboratory conditions.

Identity and quality control

• Identity inferred from molecular formula, molecular weight, and matching InChI key (e.g., InChI Key: NIPYQLPZPLBOLF‑GBLLEDPASA‑N for fluorescein‑mono‑β‑D‑galactoside).
• Quality‑control generally includes checks for appearance, solubility, spectral properties (UV‑Vis/fluorescence), and performance in a defined β‑galactosidase activity assay (e.g., microplate‑based or in situ cell‑based formats).

Shelf life and storage

• Shelf life is generally 12–24 months when stored at −20 °C, sealed in a dry, light‑protected container, often recommended for hygroscopic and light‑sensitive material.
• Aqueous working solutions are best prepared fresh or stored at 4 °C for short periods; long‑term storage, UV‑/visible‑light exposure, and elevated temperature should be avoided to prevent photodegradation or nonspecific hydrolysis.

Application

• Fluorimetric assay of β‑galactosidase activity in bacterial, fungal, plant, and mammalian systems.
• Use in reporter‑gene assays (e.g., lacZ, β‑galactosidase complementation) and in situ cell‑based imaging of β‑galactosidase activity.
• Inhibitor‑screening and enzyme‑mechanism‑elucidation studies for β‑galactosidases and related glycosidases.

Key characteristics

• Fluorogenic readout based on fluorescein release, enabling highly sensitive, microplate‑compatible detection of β‑galactosidase activity in the green‑fluorescence channel.
• Defined β‑D‑galactopyranoside linkage providing selectivity for β‑galactosidases over many α‑glycosidases.
• High‑purity, lyophilized solid with a known molecular formula and weight, suitable for quantitative enzyme assays and standardized reporter‑gene protocols.

Citations

• Fluorochem – Fluorescein mono‑β‑D‑Galactoside (FMGal, CAS 102286‑67‑9, product code M03593, formula C₂₆H₂₂O₁₀, MW 494.45): https://fluorochem.co.uk/product/M03593/
• Glentham Life Sciences – Fluorescein‑β‑D‑galactopyranoside (FMGal, CAS 102286‑67‑9, general‑product listing): https://www.glentham.com/en/products/categories/all/view‑100‑all/?q=&page=130
• Glentham (German) – Product search‑result for FMGal (CAS 102286‑67‑9, “Fluorescein di‑β‑D‑glucopyranoside” context page): https://www.glentham.com/de/products/search/?q=NG+011&page=24
• MedChemExpress – Related 4‑methylumbelliferyl‑β‑D‑galactoside (MUGal, CAS 7276‑28‑0) explanation (for methodological context on fluorogenic galactoside substrates): https://www.medchemexpress.com/4‑methylumbelliferyl‑β‑d‑galactopyranoside.html
• Sigma‑Aldrich – 4‑Methylumbelliferyl‑β‑D‑galactoside technical document (general protocol for fluorogenic glycoside assays, relevant to FMGal‑type methodologies): https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/397/748/m1633pis.pdf
• General CAS‑query listing for CAS 102286‑67‑9 (FMGal) and related microbial‑research‑reagent catalog: https://www.jm‑bio.com/search.do?a=s&searchtype=100&psize=10&q=102286‑67‑9
• Fluorescein‑based assay‑technology review (context for fluorescein‑mono‑glycosides in fluorogenic reporter systems): [https://www.ncbi.nlm.nih.gov/pmc/] (search “fluorescein‑mono‑β‑D‑galactoside β‑galactosidase assay”).
• Reporter‑gene and enzyme‑assay methodology paper using fluorogenic galactoside substrates (e.g., lacZ or β‑galactosidase‑activity imaging): https://www.ncbi.nlm.nih.gov/pubmed/ (search “fluorescein‑mono‑β‑D‑galactoside reporter”).
• Pharmaceutical‑microbiology manual (for general microbial‑contamination‑control concepts applicable to non‑sterile biochemical‑reagent handling): https://www.fda.gov/media/88801/download
• General glycosidase‑substrate catalog (FMGal and related fluorogenic galactosides overview): https://www.chemicalbook.com/ (search for “102286‑67‑9”).