3,3′-Diaminobenzidine (DAB) is an

organic compound Some chemical authorities define an organic compound as a chemical compound that contains a carbon–hydrogen or carbon–carbon bond; others consider an organic compound to be any chemical compound that contains carbon. For example, carbon-co ...

with the formula (C₆H₃(NH₂)₂)₂. This derivative of benzidine is a precursor to polybenzimidazole, which forms

fiber Fiber (spelled fibre in British English; from ) is a natural or artificial substance that is significantly longer than it is wide. Fibers are often used in the manufacture of other materials. The strongest engineering materials often inco ...

s that are renowned for their chemical and thermal stability.Hans Schwenecke, Dieter Mayer "Benzidine and Benzidine Derivatives" in ''Ullmann's Encyclopedia of Industrial Chemistry'', 2005, Wiley-VCH, Weinheim. As its water-soluble tetrahydrochloride, DAB has been used in immunohistochemical staining of

nucleic acid Nucleic acids are large biomolecules that are crucial in all cells and viruses. They are composed of nucleotides, which are the monomer components: a pentose, 5-carbon sugar, a phosphate group and a nitrogenous base. The two main classes of nuclei ...

s and

proteins Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, re ...

Structure

DAB is symmetric about the central carbon bond between both ring structures. In the crystal, the rings of each molecule are co-planar and the amine units connect molecules to form an intermolecular 3-dimensional hydrogen bond network.

Preparation

Diaminobenzidine, which is commercially available, is prepared by treating 3,3′-dichlorobenzidine with

ammonia Ammonia is an inorganic chemical compound of nitrogen and hydrogen with the chemical formula, formula . A Binary compounds of hydrogen, stable binary hydride and the simplest pnictogen hydride, ammonia is a colourless gas with a distinctive pu ...

with a copper catalyst at high temperature and pressure, followed by acidic workup. An alternate synthesis route involves the di acylation of benzidine with acetic anhydride under basic conditions: :(NH₂)C₆H₄C₆H₄(NH₂) + 2 (CH₃CO)₂O ⟶ (NHCOCH₃)C₆H₄C₆H₄(NHCOCH₃) + 2 CH₃COOH The diacetylated compound then undergoes nitration with

nitric acid Nitric acid is an inorganic compound with the formula . It is a highly corrosive mineral acid. The compound is colorless, but samples tend to acquire a yellow cast over time due to decomposition into nitrogen oxide, oxides of nitrogen. Most com ...

to produce an ortho-dinitro compound due to the ortho-directing acetyl substituents: :(NHCOCH₃)C₆H₄C₆H₄(NHCOCH₃) + 2HNO₃ ⟶ (O₂N)(NHCOCH₃)C₆H₃C₆H₃(NHCOCH₃)(NO₂) + 2H₂O The acetyl groups are then removed through saponification: :(O₂N)(NHCOCH₃)C₆H₃C₆H₃(NHCOCH₃)(NO₂) + 2NaOH ⟶ (O₂N)(NH₂)C₆H₃C₆H₃(NH₂)(NO₂) + 2NaOCOCH₃ The dinitrobenzidine compound is then reduced with hydrochloric acid and

iron Iron is a chemical element; it has symbol Fe () and atomic number 26. It is a metal that belongs to the first transition series and group 8 of the periodic table. It is, by mass, the most common element on Earth, forming much of Earth's o ...

to produce 3,3′-diaminobenzidine: :(O₂N)(NH₂)C₆H₃C₆H₃(NH₂)(NO₂) + 12HCl + 6Fe⁰ ⟶ (NH₂)₂C₆H₃C₆H₃(NH₂)₂ + 6FeCl₂ + 4H₂O The reduction of the dinitrobenzidine compound can also proceed with tin(II) chloride instead of iron powder or with sodium dithionite in methanol.

Applications

In its main application, DAB is the precursor to polybenzimidazole. Diaminobenzidine is oxidized by

hydrogen peroxide Hydrogen peroxide is a chemical compound with the formula . In its pure form, it is a very pale blue liquid that is slightly more viscosity, viscous than Properties of water, water. It is used as an oxidizer, bleaching agent, and antiseptic, usua ...

in the presence of

hemoglobin Hemoglobin (haemoglobin, Hb or Hgb) is a protein containing iron that facilitates the transportation of oxygen in red blood cells. Almost all vertebrates contain hemoglobin, with the sole exception of the fish family Channichthyidae. Hemoglobin ...

to give a dark-brown color. This color change is used to detect fingerprints in blood. The solubility of DAB in water allows for adaptability compared to other detection solutions which use toxic solvents. Improperly prepared tissue samples may give false positives. In research, this reaction is used to stain cells that were prepared with hydrogen peroxidase enzyme, following common immunocytochemistry protocols. Relevant to

Alzheimer's disease Alzheimer's disease (AD) is a neurodegenerative disease and the cause of 60–70% of cases of dementia. The most common early symptom is difficulty in remembering recent events. As the disease advances, symptoms can include problems wit ...

, Aβ protein amyloid plaques are targeted by a primary antibody, and subsequently by a secondary antibody, which is conjugated with a peroxidase enzyme. This will bind DAB as a substrate and oxidize it, producing an easily observable brown color. Plaques can then be quantified for further evaluation. One other method uses complexes of injected biocytin with

avidin Avidin is a tetrameric biotin-binding protein produced in the oviducts of birds, reptiles and amphibians and deposited in the whites of their eggs. Dimeric members of the avidin family are also found in some bacteria. In chicken egg white, a ...

or streptavidin, biotin, and then peroxidase.

References

External links

Fishersci.com

Polysciences.com
{{DEFAULTSORT:Diaminobenzidine, 3,3'- Anilines Biphenyls