fluorogenic substrate; phosphatase
The target of DiFMUP is not a biological receptor but rather the active site of phosphatase enzymes. It serves as a substrate that is dephosphorylated by these enzymes, releasing the highly fluorescent product. Its affinity for phosphatases is determined by the enzyme's catalytic efficiency. The compound is a research tool used to monitor phosphatase activity in high-throughput screening and kinetic studies.

Protein tyrosine phosphatase 1B (PTP1B) hydrolyzes DIFMUP (100 μM; 0–4 min) in a time-dependent manner, with the initial rate of reaction rising as the enzyme concentration increases (30–600 ng/mL) [2].

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In vitro, DiFMUP is used in a variety of assays to measure phosphatase activity. Its fluorescence properties allow for continuous monitoring of enzyme kinetics in real time. The excitation and emission maxima are approximately 358 nm and 450 nm, respectively. The compound is stable at physiological pH and is compatible with many biological buffers. Its high sensitivity enables detection of low enzyme concentrations and is suitable for screening inhibitors.

In vivo, DiFMUP is not intended for use as a therapeutic agent. Its application is strictly in vitro for enzymatic assays. However, it can be used in cell-based assays where cellular phosphatases are released or accessed, such as in cell lysis assays or in situ phosphatase activity detection. It is not administered to live animals.

For determination of the appropriate enzyme concentration, PTPs were prediluted in reaction buffer to the indicated concentrations in a final volume of 90 μl. Reactions were initiated by adding 10 μl containing 1 mM DIFMUP and then incubated at 37 °C while monitoring the hydrolysis for 10 min.[2]

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In vitro enzyme assays with DiFMUP involve incubating the substrate with the phosphatase enzyme of interest in a suitable buffer. The reaction is monitored by measuring the increase in fluorescence at 450 nm upon excitation at 358 nm. The initial rate of fluorescence increase is proportional to enzyme activity. For inhibition studies, the compound is used at a fixed concentration (e.g., 10-100 µM) and inhibitors are added to the reaction mixture.

In vitro cell-based assays using DiFMUP typically involve treating cells with a lysis buffer that releases intracellular phosphatases. The lysate is then incubated with DiFMUP, and fluorescence is measured to quantify phosphatase activity. Alternatively, live cells can be incubated with the substrate if the cell membrane is permeabilized. The assay is compatible with 96-well plate formats and is used for drug screening.

In vivo animal studies are not applicable for DiFMUP as it is a biochemical reagent. However, if used in ex vivo tissue homogenates, the compound can be incubated with tissue extracts to measure total phosphatase activity. The protocols involve homogenizing tissues in appropriate buffers, centrifuging, and using the supernatant for the fluorescence assay. All procedures would follow standard laboratory protocols.

The pharmacokinetic properties of DiFMUP are not relevant as it is not a drug. It is a small molecule with a molecular weight of 292.13 g/mol, soluble in water and DMSO. It is stable when stored at -20°C, protected from light. For research use, it is typically dissolved in DMSO as a stock solution and diluted in aqueous buffers.

The toxicity profile of DiFMUP is not well characterized because it is used only in vitro. As a non-toxic probe, it is generally considered safe for laboratory handling. However, standard safety precautions for handling chemicals should be followed, including wearing gloves and eye protection. It is not intended for human consumption.

[1]. Fluorogenic substrates based on fluorinated umbelliferones for continuous assays of phosphatases and beta-galactosidases. Anal Biochem. 1999 Aug 15;273(1):41-8.

[2]. 6,8-Difluoro-4-methylumbiliferyl phosphate: a fluorogenic substrate for protein tyrosine phosphatases. Anal Biochem. 2005 Mar 1;338(1):32-8.

6,8-Difluoro-4-methylumbelliferone phosphate is a coumarin compound with functions similar to coumarins. It is the conjugate acid of 6,8-difluoro-4-methylumbelliferone phosphate (2-). Fluorescent substrates based on 4-methylumbelliferone (4-MU) have been widely used to detect the activity of phosphatases and glycosidases. However, a drawback of these substrates is that, due to the pKa value of 4-MU being approximately 8, the maximum fluorescence intensity of the reaction products requires alkaline pH conditions. After initial screening of five phosphatase substrates based on fluorinated derivatives of 4-MU (all with pKa values lower than 4-MU), we found that one substrate, 6,8-difluoro-4-methylumbelliferone phosphate (DiFMUP), significantly improved the detection of acid phosphatase activity. At the optimal pH (5.0) for the acid phosphatase reaction, DiFMUP produced a fluorescence signal more than 10 times higher than that of 4-methylumbelliferone phosphate (MUP). At pH 7, DiFMUP was superior to MUP in detecting protein phosphatase 1 activity; while at pH 10, the sensitivity of DiFMUP in detecting alkaline phosphatase activity was comparable to that of MUP. In addition, a β-galactosidase substrate was prepared based on 6,8-difluoro-4-methylumbelliferone. The substrate 6,8-difluoro-4-methylumbelliferone β-D-galactopyranoside (DiFMUG) showed significantly higher sensitivity than the commonly used substrate 4-methylumbelliferone β-D-galactopyranoside (MUG) in detecting β-galactosidase activity at pH 7. DiFMUP and DiFMUG have broad application prospects in the continuous determination of phosphatases and β-galactosidase activities under neutral and acidic pH conditions, respectively. [1]

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The fluorescent substrate 6,8-difluoro-4-methylumbelliferone phosphate (DIFMUP) has been widely used to detect the activity of serine and threonine phosphatases. This article will introduce the application of this substrate in the characterization of protein tyrosine phosphatases (PTPs) and the screening of PTP inhibitors. The kinetic constants and inhibitory constants of DIFMUP cleavage are comparable to those of widely used but less selective and practical substrates—p-nitrophenyl phosphate and phosphotyrosine-containing peptides. Furthermore, this continuous and highly sensitive detection method can rapidly and accurately study the type, kinetic behavior, and binding mode of small molecule inhibitors. We discuss the validation of this detection system for various PTPs and its application in the screening of PTP1B inhibitors. [2]

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Additional information: DiFMUP is also known as 6,8-difluoro-4-methylumbelliferyl phosphate, disodium salt. It is a popular choice for fluorogenic phosphatase assays due to its improved performance over the traditional 4-methylumbelliferyl phosphate (MUP). The product is for research use only and is not approved for diagnostic or therapeutic applications.

C10H7O6F2P

292.12958

291.995

C, 41.12; H, 2.42; F, 13.01; O, 32.86; P, 10.60

214491-43-7

2786976

White to off-white solid

1.851

2

8

2

19

461

0

FC1=CC2C(C)=CC(=O)OC=2C(F)=C1OP(O)(O)=O

DZANYXOTJVLAEE-UHFFFAOYSA-N

InChI=1S/C10H7F2O6P/c1-4-2-7(13)17-9-5(4)3-6(11)10(8(9)12)18-19(14,15)16/h2-3H,1H3,(H2,14,15,16)

(6,8-Difluoro-4-methyl-2-oxochromen-7-yl) dihydrogen phosphate

DiFMUP; 214491-43-7; 6,8-difluoro-4-methyl-umbelliferyl phosphate; 6,8-difluoro-4-methylumbelliferyl phosphate; (6,8-difluoro-4-methyl-2-oxochromen-7-yl) dihydrogen phosphate; 6,8-difluoro-MUP; SCHEMBL329996; BDBM13432;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~171.16 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)