Fluorescein diacetate
[CAS# 596-09-8]

Identification

• Classification: Analytical chemistry >> Standard >> Pharmacopoeia standards and magazine standards
• Name: Fluorescein diacetate
• Synonyms: 3-Oxospiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3',6'-diyl diacetate
• Molecular Formula: C₂₄H₁₆O₇
• Molecular Weight: 416.38
• CAS Registry Number: 596-09-8
• EC Number: 209-877-6
• SMILES: CC(=O)OC1=CC2=C(C=C1)C3(C4=C(O2)C=C(C=C4)OC(=O)C)C5=CC=CC=C5C(=O)O3

Properties

• Density: 1.5±0.1 g/cm³ Calc.^*
• Melting point: 200 - 203 °C (Expl.)
• Boiling point: 604.7±55.0 °C 760 mmHg (Calc.)^*
• Flash point: 264.0±31.5 °C (Calc.)^*
• Solubility: DMSO:48mg/mL (Expl.)
• Index of refraction: 1.681 (Calc.)^*

^* Calculated using Advanced Chemistry Development (ACD/Labs) Software.

Safety Data

• Hazard Symbols: GHS07 Warning
• Risk Statements: H302-H315-H319-H335
• Safety Statements: P261-P305+P351+P338

Discovery and Applications

Fluorescein diacetate is an acetylated derivative of fluorescein with the molecular formula C₂₄H₁₆O₈. Structurally, it consists of a fluorescein core in which the two hydroxyl groups at the 3′ and 6′ positions are converted to acetate esters. This modification renders the molecule non-fluorescent and more hydrophobic compared to the parent fluorescein, enhancing its ability to permeate cell membranes. Fluorescein diacetate typically appears as a pale yellow solid that is soluble in organic solvents such as acetone, ethanol, and dimethyl sulfoxide, while its solubility in water is limited until hydrolyzed.

The compound is widely used as a fluorogenic probe in cell biology and enzymology. Once inside viable cells, intracellular esterases hydrolyze the acetate groups to regenerate fluorescein, which is strongly fluorescent under appropriate excitation. This property enables its use in assays to measure cell viability, esterase activity, and membrane integrity. Cells that are metabolically active convert fluorescein diacetate to fluorescent fluorescein, whereas dead or compromised cells remain non-fluorescent.

Fluorescein diacetate is also applied in flow cytometry, fluorescence microscopy, and high-throughput screening assays to quantify live cell populations, monitor enzyme activity, or track intracellular processes. Its hydrophobic diacetate form allows efficient cellular uptake, while enzymatic cleavage produces a water-soluble, charged fluorescent product that is retained within cells.

Synthetically, fluorescein diacetate is prepared by acetylation of fluorescein using acetic anhydride or acetyl chloride under basic or catalytic conditions. The reaction selectively converts the hydroxyl groups to acetate esters while preserving the xanthene core responsible for fluorescence after hydrolysis. Handling precautions include avoiding exposure to strong acids or bases that could prematurely hydrolyze the esters, as well as standard practices for light-sensitive fluorescent compounds.

Overall, fluorescein diacetate is a valuable fluorogenic compound for biological and biochemical research. Its cell-permeable diacetate form and enzymatically generated fluorescent product make it a widely used tool for assessing cell viability, esterase activity, and other intracellular processes, providing reliable and sensitive detection in vitro and in live-cell studies.

References

2007. Single Cell Level Microalgal Ecotoxicity Assessment by Confocal Microscopy and Digital Image Analysis. Environmental Science & Technology, 41(9).
DOI: 10.1021/es0627390

2010. Development and Validation of a Quantitative, High-Throughput, Fluorescent-Based Bioassay to Detect Schistosoma Viability. PLoS Neglected Tropical Diseases, 4(7).
DOI: 10.1371/journal.pntd.0000759