3,3′-Diaminobenzidine (DAB) is an

organic compound In chemistry, organic compounds are generally any chemical compounds that contain carbon-hydrogen or carbon-carbon bonds. Due to carbon's ability to catenate (form chains with other carbon atoms), millions of organic compounds are known. The ...

with the formula (C₆H₃(NH₂)₂)₂. This derivative of

benzidine Benzidine (trivial name), also called 1,1'-biphenyl-4,4'-diamine (systematic name), is an organic compound with the formula (C6H4NH2)2. It is an aromatic amine. It is a component of a test for cyanide. Related derivatives are used in the produc ...

is a precursor to

polybenzimidazole Polybenzimidazole (PBI, short for poly ,2’-(''m''-phenylen)-5,5’-bisbenzimidazole'') fiber is a synthetic fiber with a very high decomposition temperature. It does not exhibit a melting point, it has exceptional thermal and chemical stability ...

, which forms

fiber Fiber or fibre (from la, fibra, links=no) 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 incorporate ...

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 Immunohistochemistry (IHC) is the most common application of immunostaining. It involves the process of selectively identifying antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to ant ...

nucleic acid Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main cl ...

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, respo ...

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 compound of nitrogen and hydrogen with the formula . A stable binary hydride, and the simplest pnictogen hydride, ammonia is a colourless gas with a distinct pungent smell. Biologically, it is a common nitrogenous was ...

with a copper catalyst at high temperature and pressure, followed by acidic workup. An alternate synthesis route involves the di acylation of

with

acetic anhydride Acetic anhydride, or ethanoic anhydride, is the chemical compound with the formula (CH3CO)2O. Commonly abbreviated Ac2O, it is the simplest isolable anhydride of a carboxylic acid and is widely used as a reagent in organic synthesis. It is a col ...

under basic conditions: :(NH₂)C₆H₄C₆H₄(NH₂) + 2 (CH₃CO)₂O ⟶ (NHCOCH₃)C₆H₄C₆H₄(NHCOCH₃) + 2 CH₃CO₂H The diacetylated compound then undergoes nitration with

nitric acid Nitric acid is the inorganic compound with the formula . It is a highly corrosive mineral acid. The compound is colorless, but older samples tend to be yellow cast due to decomposition into oxides of nitrogen. Most commercially available nitri ...

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 In organic chemistry, acetyl is a functional group with the chemical formula and the structure . It is sometimes represented by the symbol Ac (not to be confused with the element actinium). In IUPAC nomenclature, acetyl is called ethanoyl, ...

groups are then removed through saponification: :(O₂N)(NHCOCH₃)C₆H₃C₆H₃(NHCOCH₃)(NO₂) + 2NaOH ⟶ (O₂N)(NH₂)C₆H₃C₆H₃(NH₂)(NO₂) + 2(NaOCOCH₃) The dinitrobenzidine compound is then reduced with

hydrochloric acid Hydrochloric acid, also known as muriatic acid, is an aqueous solution of hydrogen chloride. It is a colorless solution with a distinctive pungent smell. It is classified as a strong acid Acid strength is the tendency of an acid, symbol ...

and

iron Iron () is a chemical element with symbol Fe (from la, ferrum) 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, right in f ...

to produce 3,3′-diaminobenzidine: :3(O₂N)(NH₂)C₆H₃C₆H₃(NH₂)(NO₂) + 12HCl + 10Fe⁰ ⟶ 3(NH₂)₂C₆H₃C₆H₃(NH₂)₂ + 4Fe₂O₃ + 6FeCl₂ The reduction of the dinitrobenzidine compound can also proceed with tin(II) chloride instead of iron powder or with

sodium dithionite Sodium dithionite (also known as sodium hydrosulfite) is a white crystalline powder with a sulfurous odor. Although it is stable in dry air, it decomposes in hot water and in acid solutions. Structure The structure has been examined by Raman s ...

methanol Methanol (also called methyl alcohol and wood spirit, amongst other names) is an organic chemical and the simplest aliphatic alcohol, with the formula C H3 O H (a methyl group linked to a hydroxyl group, often abbreviated as MeOH). It is a ...

Applications

In its main application, DAB is the precursor to

. 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 viscous than water. It is used as an oxidizer, bleaching agent, and antiseptic, usually as a dilute solution (3%� ...

in the presence of

hemoglobin Hemoglobin (haemoglobin BrE) (from the Greek word αἷμα, ''haîma'' 'blood' + Latin ''globus'' 'ball, sphere' + ''-in'') (), abbreviated Hb or Hgb, is the iron-containing oxygen-transport metalloprotein present in red blood cells (erythrocyte ...

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 Immunocytochemistry (ICC) is a common laboratory technique that is used to anatomically visualize the localization of a specific protein or antigen in cells by use of a specific primary antibody that binds to it. The primary antibody allows visual ...

protocols. Relevant to

Alzheimer's disease Alzheimer's disease (AD) is a neurodegeneration, neurodegenerative disease that usually starts slowly and progressively worsens. It is the cause of 60–70% of cases of dementia. The most common early symptom is difficulty in short-term me ...

, Aβ protein

amyloid plaques Amyloid plaques (also known as neuritic plaques, amyloid beta plaques or senile plaques) are extracellular deposits of the amyloid beta (Aβ) protein mainly in the grey matter of the brain. Degenerative neuronal elements and an abundance of micr ...

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 Biocytin is a chemical compound that is an amide formed from the vitamin biotin and the amino acid L-lysine. As an intermediate in the metabolism of biotin, biocytin occurs naturally in blood serum and urine. The enzyme biotinidase cleaves bioc ...

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, avidin ...

streptavidin Streptavidin is a 66.0 (tetramer) kDa protein purified from the bacterium '' Streptomyces avidinii''. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin (also known as vitamin B7 or vitamin H). With a dissociation co ...

, biotin, and then peroxidase.

References

External links

Fishersci.com

Polysciences.com
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