4-Nitrophenyl phosphate (p-nitrophenyl phosphate) disodium hexahydrate is extensively used as a small molecule phosphotyrosine-like substrate in protein tyrosine phosphatase activity assays. 4-Nitrophenyl phosphate disodium hexahydrate is a colorless matrix that is converted to yellow 4-nitrophenol ions after hydrolysis, which can be monitored by absorbance at 405 nm.

Physicochemical Properties

Molecular Formula	C6H16NNA2O12P
Molecular Weight	371.14
Exact Mass	371.021
CAS #	333338-18-4
Related CAS #	4-Nitrophenyl phosphate ditromethamine;68189-42-4
PubChem CID	2723710
Appearance	Off-white to light yellow solid powder
Boiling Point	457.8ºC at 760mmHg
Melting Point	>300ºC(lit.)
Flash Point	230.7ºC
LogP	2.08
Hydrogen Bond Donor Count	6
Hydrogen Bond Acceptor Count	12
Rotatable Bond Count	1
Heavy Atom Count	22
Complexity	238
Defined Atom Stereocenter Count	0
InChi Key	KAKKHKRHCKCAGH-UHFFFAOYSA-L
InChi Code	InChI=1S/C6H6NO6P.2Na.6H2O/c8-7(9)5-1-3-6(4-2-5)13-14(10,11)12;;;;;;;;/h1-4H,(H2,10,11,12);;;6*1H2/q;2*+1;;;;;;/p-2
Chemical Name	disodium;(4-nitrophenyl) phosphate;hexahydrate
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vitro	4-Nitrophenyl phosphate (PNPP) disodium hexahydrate is a substrate that alkaline phosphatases (ALPs) frequently employ. 4-ALP hydrolyzes nitrophenyl phosphate disodium hexahydrate to PNP (p-nitrophenol), which uses the inner filter effect (IFE) to dim the fluorescence of new gold nanoclusters (AuNCs)[2].
References	[1]. Structural analysis of human dual-specificity phosphatase 22 complexed with a phosphotyrosine-like substrate. Acta Crystallogr F Struct Biol Commun. 2015;71(Pt 2):199-205. [2]. Development of a facile and sensitive method for detecting alkaline phosphatase activity in serum with fluorescent gold nanoclusters based on the inner filter effect. Analyst. 2020;145(11):3871-3877.
Additional Infomation	A group of hydrolases which catalyze the hydrolysis of monophosphoric esters with the production of one mole of orthophosphate.

Solubility Data

Solubility (In Vitro)

H2O: 100 mg/mL (269.44 mM) DMSO: 10 mg/mL (26.94 mM)

Solubility (In Vivo)

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. Injection Formulations (e.g. IP/IV/IM/SC) 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). 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) Oral Formulations 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). 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 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.6944 mL	13.4720 mL	26.9440 mL
	5 mM	0.5389 mL	2.6944 mL	5.3888 mL
	10 mM	0.2694 mL	1.3472 mL	2.6944 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.