4-Nitrophenyl phosphate ditromethamine
[CAS 68189-42-4]

Identification

• Classification: Organic raw materials >> Carboxylic compounds and derivatives >> Salt of carboxylic acid ester and its derivatives
• Name: 4-Nitrophenyl phosphate ditromethamine
• Synonyms: bis(2-amino-2-(hydroxymethyl)propane-1,3-diol), (4-nitrophenyl) dihydrogen phosphate
• Molecular Formula: C₆H₆NO₆P .₂C₄H₁₁NO₃
• Molecular Weight: 461.36
• CAS Registry Number: 68189-42-4
• EC Number: 269-198-6
• SMILES: C1=CC(=CC=C1[N+](=O)[O-])OP(=O)(O)O.C(C(CO)(CO)N)O.C(C(CO)(CO)N)O

Safety Data

• Hazard Symbols: GHS07 Warning
• Risk Statements: H302
• Safety Statements: P264-P270-P301+P317-P330-P501

Hazard Classification

Hazard	Class	Category Code	Hazard Statement
Acute toxicity	Acute Tox.	4	H302

Discovery and Applications

4-Nitrophenyl phosphate ditromethamine is a salt form of 4-nitrophenyl phosphate (pNPP) in which the phosphate-containing compound is associated with two equivalents of tromethamine (tris(hydroxymethyl)aminomethane, often abbreviated Tris). It is commonly used as a chromogenic substrate in biochemical and enzymatic assays, particularly for the measurement of phosphatase activity.

The central component, 4-nitrophenyl phosphate, consists of a phosphate ester attached to a para-substituted nitrophenyl ring. The aromatic ring contains a nitro group (–NO₂) at the 4-position relative to the phosphate ester linkage. The nitro group is strongly electron-withdrawing and significantly influences the electronic properties of the aromatic system.

The phosphate group is highly polar and can exist in ionized forms depending on pH. In the ditromethamine salt form, negatively charged phosphate oxygens are stabilized by protonated tromethamine molecules through ionic interactions. Tromethamine itself contains a primary amine and three hydroxymethyl groups, giving it buffering capacity and high water compatibility.

The most well-established application of 4-nitrophenyl phosphate is as a substrate for phosphatase enzymes, especially alkaline phosphatase and acid phosphatase assays. During enzymatic hydrolysis, the phosphate ester bond is cleaved to produce inorganic phosphate and 4-nitrophenol. Under alkaline conditions, 4-nitrophenol is converted to its deprotonated form, 4-nitrophenolate, which exhibits a characteristic yellow color. This color development allows enzyme activity to be monitored spectrophotometrically.

Because the reaction product absorbs light strongly in the visible region, pNPP-based assays have become widely used in biochemical analysis, enzyme kinetics studies, immunoassays, and diagnostic applications. The chromogenic nature of the substrate makes quantitative analysis relatively straightforward.

From a physicochemical standpoint, the aromatic nitrophenyl portion contributes hydrophobic character, whereas the phosphate and tromethamine components provide substantial polarity and hydrogen-bonding capability. The salt form improves aqueous solubility compared with less ionized forms of the compound.

Chemically, the phosphate ester linkage is the principal reactive site. Hydrolysis of this bond, either enzymatically or under sufficiently strong chemical conditions, yields the corresponding phenol and phosphate products. The nitro-substituted aromatic ring itself is generally stable under normal handling conditions.

Overall, 4-nitrophenyl phosphate ditromethamine is best described as a water-compatible salt form of a phosphate ester substrate whose importance lies primarily in its established use as a chromogenic reagent for phosphatase detection and measurement in biochemical research and analytical applications.