5-Acetamidofluorescein-di-(β-D-galactopyranoside)

5-Acetamidofluorescein-di-(β-D-galactopyranoside) (C2FDG, CAS 216299-45-5) is a cell-permeant fluorogenic bis-substrate specifically designed for highly sensitive detection of β-galactosidase (lacZ) activity in live cells, microbial systems, and gene expression studies. The non-fluorescent molecule features two β-D-galactopyranoside moieties masking the 5-acetamido fluorescein fluorophore; sequential enzymatic hydrolysis releases mono-galactosyl and ultimately free 5-acetamido fluorescein (λex 485 nm, λem 520 nm), producing bright green fluorescence proportional to enzyme activity for quantitative flow cytometry, microscopy, and high-throughput screening. This acetamido modification enhances aqueous solubility and cellular retention compared to FDG (CAS 17817-20-8), making it ideal for real-time monitoring of β-galactosidase in bacterial reporter strains, viral transduction efficiency, senescence-associated β-gal (SA-β-gal) assays, and living cell promoter studies with minimal toxicity and high signal-to-noise ratios.

Appearance

• Off-white to pale yellow amorphous solid or lyophilized powder.
• Highly soluble in water/DMSO (>10 mM); stable clear solutions.

Source

• Multi-step organic synthesis: 5-acetamido fluorescein glycosylated with protected β-D-galactopyranosyl donors via Lewis acid catalysis, followed by deprotection.
• Commercial production for cell biology research (Biosynth EA03178, Apollo Scientific BICL4232).

Molecular Weight and Structure

• C34H35NO16, 713.65 g/mol; exact mass 713.1975 Da.
• 5-(acetylamino)-3′,6′-bis(β-D-galactopyranosyloxy)spiro[isobenzofuran-1(3H),9′-[9H]xanthene]-3-one.

Sugar Specificity

• Specific for β-galactosidases (EC 3.2.1.23); sequential hydrolysis of both Gal-β(1→O)-fluorescein bonds.
• Km1 ≈ 30-50 μM, Km2 ≈ 5-10 μM; minimal cross-reactivity with other glycosidases.

Biological Activity

• β-Galactosidase hydrolyzes → 5-acetamido-fluorescein mono-β-Gal + Gal, then free fluorophore (λex/λem 488/520 nm).
• Fluorescence increase 1000-fold; optimal pH 6.0-7.4, 37°C; linear to 1 mU/mL enzyme.

Purity and Microbial Contamination

• ≥98% HPLC purity; single peak; free fluorescein <0.1%.
• Endotoxin-free (<0.05 EU/mg); sterile DMSO stocks for cell culture.

Identity and Quality Control

• Confirmed by LC-MS (m/z 714.2 [M+H]+), 1H-NMR (Gal H-1 doublet J=7.8 Hz), fluorescence titration assay.
• Functional test: >500-fold fluorescence enhancement with E. coli lacZ.

Shelf Life and Storage

• 24 months at -20°C as 10-50 mM DMSO stock; lyophilized powder >36 months.
• Protect from light/moisture; avoid repeated freeze-thaws.

Application

• Live-cell lacZ reporter quantification by flow cytometry/microscopy.
• SA-β-gal senescence assays; viral transduction efficiency; HTS gene expression.

Key Characteristics

• Cell membrane permeant; 10x brighter than FDG; low toxicity (<10 μM).
• Real-time kinetic measurement; ratiometric potential; 488 nm laser compatible.

Citation Links

• https://store.apolloscientific.co.uk/product/5-acetamidofluorescein-di-beta-d-galactopyranoside
• https://www.chemicalbook.com/ChemicalProductProperty_EN_CB21231715.htm
• https://pmc.ncbi.nlm.nih.gov/articles/PMC12222868/
• https://www.biosynth.com/p/EA03178/216299-45-5-5-acetamidofluorescein-di-b-d-galact
• https://www.abcam.com/en-us/products/biochemicals/fluorescein-di-d-galactopyranoside-fdg-ab273643
• https://www.semanticscholar.org/paper/eaf81d9816025cfe874963750db079636b752727
• https://www.chemsrc.com/en/cas/17817-20-8_606553.html
• https://www.biosynth.com/category/fluorogenic-substrates/view-all
• https://www.evitachem.com/product/evt-1473482
• https://www.merckmillipore.com/BO/en/product/sigma/f2756