2-[[[(5-Bromo-4-chloro-1H-indol-3-yl)oxy]hydroxyphosphinyl]oxy]-N,N,N-trimethylethanaminium inner salt
[CAS# 439809-43-5]

Identification

• Classification: Organic raw materials >> Heterocyclic compound >> Indoles
• Name: 2-[[[(5-Bromo-4-chloro-1H-indol-3-yl)oxy]hydroxyphosphinyl]oxy]-N,N,N-trimethylethanaminium inner salt
• Synonyms: 5-Bromo-4-chloro-3-indoxyl choline phosphate
• Molecular Formula: C₁₃H₁₇BrClN₂O₄P
• Molecular Weight: 411.62
• CAS Registry Number: 439809-43-5
• SMILES: C[N+](C)(C)CCOP(=O)([O-])OC1=CNC2=C1C(=C(C=C2)Br)Cl

Safety Data

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

Discovory and Applicatios

2-$[[(5-Bromo-4-chloro-1H-indol-3-yl)oxy$hydroxyphosphinyl]oxy]-N,N,N-trimethylethanaminium inner salt is a synthetic organophosphorus compound used as a chromogenic substrate for detecting alkaline phosphatase (AP) activity in histochemical and immunoassay applications. This molecule is commonly abbreviated as BCIP (5-Bromo-4-chloro-3-indolyl phosphate), and in its most widely used form, it is coupled with NBT (nitro blue tetrazolium) to form the BCIP/NBT substrate system, enabling high-sensitivity detection in colorimetric assays.

The development of BCIP was part of broader efforts in the 20th century to create substrates for enzyme-based detection systems. Alkaline phosphatase, an enzyme found in many tissues and used extensively as a reporter in immunological and molecular assays, catalyzes the hydrolysis of phosphate esters. Researchers sought stable, non-toxic substrates that could produce insoluble and visually detectable products upon enzymatic cleavage. The BCIP molecule was designed with a phosphate ester group attached to a substituted indole structure, allowing it to undergo enzymatic hydrolysis followed by oxidation to yield a chromogenic product.

Upon reaction with alkaline phosphatase, the phosphate group in BCIP is cleaved, producing the intermediate 5-bromo-4-chloro-3-indoxyl. This intermediate is then oxidized in the presence of oxygen, or preferably in combination with an oxidizing partner such as NBT, to form an insoluble purple precipitate consisting primarily of 5,5'-dibromo-4,4'-dichloro-indigo and reduced NBT (formazan). This insoluble colored product precipitates at the site of enzyme activity, enabling precise localization of AP in tissue sections, membranes, or microplate wells.

The use of BCIP has become standard in non-radioactive detection methods, especially in Western blotting, Southern blotting, and in situ hybridization. In these techniques, antibodies or nucleic acid probes are labeled with alkaline phosphatase, and BCIP is used as the chromogenic substrate. The formation of the purple precipitate signals the presence of the target antigen or nucleic acid sequence. This provides a visible and permanent record of the detection event, which can be archived and reviewed.

In histochemical applications, BCIP is used to visualize alkaline phosphatase activity in tissue samples. It is applied in conjunction with NBT in buffered solutions that maintain the enzyme's activity. Tissue sections incubated with the substrate system develop intense purple coloration in regions expressing alkaline phosphatase. This is useful for mapping enzyme distribution in normal and pathological conditions, including studies of liver, intestine, kidney, and bone tissues, where AP is physiologically active.

The BCIP/NBT system is also widely used in immunocytochemistry and immunohistochemistry, where it serves as a chromogenic detection system in assays involving labeled antibodies. One of its advantages is its high sensitivity and the stability of the color precipitate, which does not diffuse and provides high-resolution staining. Additionally, the system does not require the use of radioactive materials, offering a safer alternative for both research and diagnostic purposes.

The compound is typically supplied as a powder or in solution and requires protection from light to prevent degradation. Assays using BCIP are generally performed at alkaline pH (around pH 9.5), which is optimal for alkaline phosphatase activity. The choice of buffer and presence of metal ions or chelators can significantly affect enzyme activity and substrate stability.

Due to its effectiveness, 2-$[[(5-Bromo-4-chloro-1H-indol-3-yl)oxy$hydroxyphosphinyl]oxy]-N,N,N-trimethylethanaminium inner salt remains a crucial reagent in molecular biology, histology, and clinical diagnostics. Its role in enabling precise, permanent, and non-radioactive detection continues to support a wide range of biological and biomedical research applications.