4-Methylumbelliferyl phosphate
[CAS# 3368-04-5]

Identification

• Classification: Pharmaceutical intermediate >> Heterocyclic compound intermediate >> Pyran compound
• Name: 4-Methylumbelliferyl phosphate
• Synonyms: 4-Methyl-2-oxo-2H-1-benzopyran-7-yl phosphate
• Molecular Formula: C₁₀H₉O₆P
• Molecular Weight: 256.15
• CAS Registry Number: 3368-04-5
• EC Number: 222-137-7
• SMILES: CC1=CC(=O)OC2=C1C=CC(=C2)OP(=O)(O)O

Properties

• Density: 1.6±0.1 g/cm³ Calc.^*
• Melting point: 215-218 °C
• Boiling point: 513.6±60.0 °C 760 mmHg (Calc.)^*
• Flash point: 264.4±32.9 °C (Calc.)^*
• Index of refraction: 1.628 (Calc.)^*

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

Safety Data

• Hazard Symbols: GHS07 Warning
• Risk Statements: H315-H319-H335
• Safety Statements: P261-P264-P264+P265-P271-P280-P302+P352-P304+P340-P305+P351+P338-P319-P321-P332+P317-P337+P317-P362+P364-P403+P233-P405-P501

Hazard Classification

Hazard	Class	Category Code	Hazard Statement
Eye irritation	Eye Irrit.	2	H319
Skin irritation	Skin Irrit.	2	H315

Discovery and Applications

4-Methylumbelliferyl phosphate is a synthetic compound widely used as a fluorogenic substrate in biochemical and enzymatic assays. It belongs to the class of phosphate esters derived from 4-methylumbelliferone, a fluorescent coumarin derivative. The molecule consists of a 4-methylumbelliferone moiety linked through a phosphate group, which is enzymatically cleaved by phosphatases to release free 4-methylumbelliferone, a highly fluorescent product. This property makes 4-methylumbelliferyl phosphate an important tool for detecting and quantifying phosphatase activity in various biological and environmental samples.

The discovery of 4-methylumbelliferyl phosphate as a fluorogenic substrate dates back to the mid-20th century when researchers sought sensitive methods to measure enzyme activities, particularly phosphatases. Unlike colorimetric substrates, fluorescent substrates provide higher sensitivity and allow real-time monitoring of enzymatic reactions. The conversion of 4-methylumbelliferyl phosphate to 4-methylumbelliferone under enzymatic action produces a distinct blue fluorescence that can be measured with spectrofluorometers, making it suitable for various analytical techniques, including high-throughput screening and kinetic studies.

Applications of 4-methylumbelliferyl phosphate extend across clinical diagnostics, biochemical research, and environmental monitoring. In clinical laboratories, it is used to assay alkaline phosphatase and acid phosphatase activities, enzymes relevant in diagnosing bone, liver, and prostate diseases. Its sensitivity allows detection of enzyme levels even at low concentrations, aiding early diagnosis and disease monitoring. In research, the compound serves as a substrate to study phosphatase function, enzyme inhibitors, and signal transduction pathways involving phosphorylation and dephosphorylation processes.

In addition to its use with purified enzymes, 4-methylumbelliferyl phosphate is employed in cellular assays to monitor phosphatase activity within live cells. The compound’s cell-permeable nature allows researchers to investigate intracellular enzyme dynamics and assess the effects of drugs or genetic modifications on phosphatase function. Its fluorescent product facilitates visualization using fluorescence microscopy and flow cytometry, broadening its utility in cell biology.

The compound’s stability and ease of use have led to its incorporation into commercial assay kits, providing standardized protocols for enzyme activity measurement. These kits are widely used in pharmaceutical development for screening potential phosphatase inhibitors, which have therapeutic implications in diseases where abnormal phosphorylation is a factor.

While 4-methylumbelliferyl phosphate offers significant advantages, certain limitations exist, including potential interference from other phosphatases and the need for careful control of assay conditions to prevent non-enzymatic hydrolysis. Nonetheless, it remains a preferred substrate due to its strong fluorescence signal, specificity, and adaptability to various assay formats.

In summary, 4-methylumbelliferyl phosphate is a valuable fluorogenic substrate pivotal in enzymology and biomedical research. Its development enhanced the capacity to detect and quantify phosphatase activities with high sensitivity, contributing to advances in diagnostics, drug discovery, and understanding of cellular processes involving phosphorylation.

References

1973. Characterization of alkaline phosphatase in tissue sections by microspectrofluorometry. Journal of Molecular Histology, 37(5).
DOI: 10.1007/bf01012062

2022. Screening of nanobody targeting alkaline phosphatase PhoX in Microcystis and detection of the PhoX in situ by fluorescence immunoassays. Journal of Applied Phycology, 34(6).
DOI: 10.1007/s10811-022-02875-z

2023. Few-layered boron nitride nanosheet as a non-metallic phosphatase nanozyme and its application in human urine phosphorus detection. Analytical and Bioanalytical Chemistry, 416(1).
DOI: 10.1007/s00216-023-05030-w