C12FDG

C12FDG is a fluorogenic β‑galactosidase‑specific derivative of fluorescein‑di‑β‑D‑galactopyranoside (FDG), in which the fluorescein core is modified with a dodecanoyl (C12) chain, making the molecule more lipophilic. The substrate consists of a 5‑dodecanoylaminofluorescein aglycone linked via two β‑glycosidic bonds to β‑D‑galactopyranose units. When hydrolyzed by β‑galactosidase, these glycosidic bonds are cleaved to release the fluorescent dodecanoylaminofluorescein and β‑D‑galactose, with fluorescence developing only after complete deglycosylation. Due to its increased lipophilicity, C12FDG shows improved membrane permeability over the parent FDG in many cell‑based systems, making it suitable for detecting β‑galactosidase activity in living or fixed mammalian cells, including in senescence‑associated β‑gal (SA‑β‑Gal) imaging and flow‑cytometry‑based reporter‑gene assays. C12FDG is supplied as a high‑purity, lyophilized or crystalline solid, compatible with PBS‑ or buffer‑based staining protocols and standard fluorescence microscopes or cytometers.

Appearance

• Off‑white to pale yellow crystalline or lyophilized powder.
• Forms a faintly yellow solution in aqueous buffer; develops bright green fluorescence only after enzymatic hydrolysis by β‑galactosidase.

Source / synthesis

• Synthetically prepared by first modifying fluorescein at the 5‑position with a dodecanoyl‑amino side chain, then glycosylating this 5‑dodecanoylaminofluorescein with two β‑D‑galactopyranose units or protected β‑D‑galactosides, followed by deprotection and purification.
• Commercially supplied by specialty‑fluorescence‑reagent vendors (e.g., Cayman Chemical, GlpBio, Sigma‑Aldrich/TargetMol, Dojindo‑linked reagents) as a research‑grade β‑galactosidase substrate.

Molecular weight and structure

• Molecular formula: C44H55NO16C44​H55​NO16​.
• Molecular weight: ~853.9 g/mol.
• Structure: 5‑dodecanoylaminofluorescein‑di‑β‑D‑galactopyranoside, with two β‑glycosidic linkages connecting the modified fluorescein core to two D‑galactose rings in the pyranose form.

Sugar specificity

• Specifically hydrolyzed by β‑galactosidases and β‑galactoside‑cleaving enzymes, analogous to fluorescein‑di‑β‑D‑galactopyranoside (FDG).
• Used in fluorogenic assays to distinguish β‑galactosidase activity from other glycosidases, especially in mammalian‑cell and reporter‑gene systems.

Biological activity

• Serves as a fluorogenic reporter whose rate of 5‑dodecanoylaminofluorescein release under defined pH and temperature conditions reflects β‑galactosidase activity.
• Shows higher sensitivity than FDG for β‑galactosidase activity determinations in animal cells, including in senescence‑associated β‑galactosidase (SA‑β‑Gal) imaging and flow‑cytometry‑based assays.

Purity and microbial contamination

• Typically supplied at ≥95% purity (e.g., “≥95%” or “≥95% 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 and cell‑based assays under clean‑laboratory conditions.

Identity and quality control

• Identity inferred from molecular formula, molecular weight, and published SMILES/InChI‑type descriptors for 5‑dodecanoylaminofluorescein‑di‑β‑D‑galactopyranoside.
• Quality‑control includes checks for appearance, solubility, spectral properties (UV‑Vis/fluorescence), and performance in defined β‑galactosidase or SA‑β‑Gal assays (e.g., protocol‑based imaging or flow‑cytometry experiments).

Shelf life and storage

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

Application

• Fluorimetric and imaging detection of β‑galactosidase activity in mammalian cells, including reporter‑gene systems and gene expression studies.
• Senescence‑associated β‑galactosidase (SA‑β‑Gal) staining in cellular‑senescence research and aging‑related studies.
• Flow‑cytometry‑ and microplate‑based β‑galactosidase‑activity quantification in animal‑cell and bacterial systems (with or without permeabilization, depending on the system).

Key characteristics

• Lipophilic fluorogenic readout: 5‑dodecanoylaminofluorescein release enables sensitive, cell‑permeable detection of β‑galactosidase activity in the green‑fluorescence channel.
• Di‑β‑D‑galactopyranoside scaffold providing high specificity for β‑galactosidases over many α‑glycosidases.
• High‑purity, lyophilized solid with a known molecular formula and weight, optimized for cellular‑imaging and SA‑β‑Gal detection protocols.

Citations

• Cayman Chemical – C12FDG (5‑Dodecanoylaminofluorescein‑di‑β‑D‑galactopyranoside, CAS 138777‑25‑0, ≥95% purity): https://www.caymanchem.com/product/25583/c12fdg
• GlpBio – C12FDG product page (CAS 138777‑25‑0, GC43021, fluorescent substrate for β‑galactosidase): https://www.glpbio.cn/c12fdg.html
• ChemSrc – C12FDG compound page (CAS 138777‑25‑0, MF C₄₄H₅₅NO₁₆, MW 853.9, chemical and assay‑use data): https://www.chemsrc.com/en/cas/138777-25-0_424177.html
• Sigma‑Aldrich (TargetMol) – C12FDG listing (CAS 138777‑25‑0, MF C₄₄H₅₅O₁₆N₁, MW 853.92): https://www.sigmaaldrich.com/US/en/product/targetmolchemicalsinc/ta9h9a1d0e3d
• Sigma‑Aldrich (AchemBlock) – C12FDG product page (same CAS, identical molecular‑formula and weight): https://www.sigmaaldrich.com/TW/en/product/achemblock/advh9a6ecfc7
• PubChem‑linked article on FDG and C12FDG use in β‑galactosidase‑detection by flow cytometry (Plovins et al., 1994): https://pubmed.ncbi.nlm.nih.gov/7811104/
• Dojindo – β‑galactosidase‑detection‑reagents guide mentioning C12FDG as a more cell‑permeable FDG variant: https://www.dojindo.com/EUROPE/contents/Galactosidase-Detection-Reagents-Selection-Guide.html
• Thermo Fisher Scientific – C12FDG product‑citation page (5‑Dodecanoylaminofluorescein‑di‑β‑D‑galactopyranoside): https://www.thermofisher.com/order/catalog/product/D2893/citations
• MedChemExpress – FDG and C12FDG method‑overview page (context for β‑galactosidase‑fluorescence assays): https://www.medchemexpress.com/mce_publications/7811104.html
• General glycosidase‑substrate catalog (C12FDG and related fluorescent galactosides overview): https://www.chemicalbook.com/ (search for “138777‑25‑0”).