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 (C
6H
3(NH
2)
2)
2. This derivative of
benzidine 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 ...
of
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.
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
In chemistry, acylation (or alkanoylation) is the chemical reaction in which an acyl group () is added to a compound. The compound providing the acyl group is called the acylating agent.
Because they form a strong electrophile when treated with ...
of
benzidine with
acetic anhydride under basic conditions:
[
:(NH2)C6H4C6H4(NH2) + 2 (CH3CO)2O ⟶ (NHCOCH3)C6H4C6H4(NHCOCH3) + 2 CH3CO2H
The diacetylated compound then undergoes ]nitration
In organic chemistry, nitration is a general class of chemical processes for the introduction of a nitro group into an organic compound. The term also is applied incorrectly to the different process of forming nitrate esters between alcohols an ...
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
Saponification is a process of converting esters into soaps and alcohols by the action of aqueous alkali (for example, aqueous sodium hydroxide solutions). Soaps are salts of fatty acids, which in turn are carboxylic acids with long carbon chains. ...
:[
:(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 ...
in 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 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 ...
.
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
Peroxidases or peroxide reductases ( EC numberbr>1.11.1.x are a large group of enzymes which play a role in various biological processes. They are named after the fact that they commonly break up peroxides.
Functionality
Peroxidases typically ca ...
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 are targeted by a primary antibody, and subsequently by a secondary antibody, which is conjugated with a peroxidase
Peroxidases or peroxide reductases ( EC numberbr>1.11.1.x are a large group of enzymes which play a role in various biological processes. They are named after the fact that they commonly break up peroxides.
Functionality
Peroxidases typically ca ...
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 or streptavidin, biotin, and then peroxidase.
References
External links
Fishersci.com
Polysciences.com
{{DEFAULTSORT:Diaminobenzidine, 3,3'-
Anilines
Biphenyls