3,3'-Diaminobenzidine

3,3'-Diaminobenzidine

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Names
IUPAC name 3,3',4,4'-Biphenyltetramine
Other names 3,3',4,4'-Tetraamino-diphenyl
Identifiers
CAS Number	91-95-2 7411-49-6 (4HCl)
ChemSpider	6804 Y
EC Number	202-110-6
Jmol 3D model	Interactive image
RTECS number	DV8750000 DV8753000 (4HCl)
InChI InChI=1S/C12H14N4/c13-9-3-1-7(5-11(9)15)8-2-4-10(14)12(16)6-8/h1-6H,13-16H2 Y Key: HSTOKWSFWGCZMH-UHFFFAOYSA-N Y InChI=1/C12H14N4/c13-9-3-1-7(5-11(9)15)8-2-4-10(14)12(16)6-8/h1-6H,13-16H2 Key: HSTOKWSFWGCZMH-UHFFFAOYAU
SMILES c2(c1cc(N)c(N)cc1)ccc(N)c(N)c2
Properties
Chemical formula	C12H14N4 C12H18Cl4N4 (4HCl)
Molar mass	214.27 g/mol 360.11 g/mol (4HCl)
Melting point	175 to 177 °C (347 to 351 °F; 448 to 450 K) (280 °C for 4HCl.2H2O)
Vapor pressure	{{{value}}}
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
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Infobox references

3,3'-Diaminobenzidine (DAB) is an organic compound that is both chemically and thermodynamically stable. This derivative of benzidine is a precursor to polybenzimidazole fiber. This fiber can be used to construct materials that are resistant to chemicals and high temperature. Fabrics made from these fibers do not melt when exposed to flame and do not burn. As its water-soluble tetrahydrochloride, DAB has been used in immunohistochemical staining of nucleic acids and proteins.

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 reacting 3,3'-dichlorobenzidine and ammonia with a copper catalyst at high temperature and pressure, followed by acidic workup.

An alternate synthesis route involves the diacylation of benzidine with acetic anhydride under basic conditions:

(NH₂)C₆H₄C₆H₄(NH₂) + 2(CH₃CO)₂O ⟶ (NHCOCH₃)C₆H₄C₆H₄(NHCOCH₃) + 2CH₃CO₂H

The diacetylated compound then undergoes nitration with nitric acid 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) + 2(NaOCOCH₃)

The dinitrobenzidine compound is then reduced with hydrochloric acid and iron as a catalyst 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 in methanol.

Applications

Diaminobenzidine is oxidized by hydrogen peroxide in the presence of hemoglobin to give a dark-brown color. This 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. In research being done on Alzheimer's disease, 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 or streptavidin, biotin, and then peroxidase.

External links

• Fishersci.com
• Polysciences.com

• Hans Schwenecke, Dieter Mayer "Benzidine and Benzidine Derivatives" in Ullmann’s Encyclopedia of Industrial Chemistry, 2005, Wiley-VCH, Weinheim.
• US 3943175, Druin, Melvin L. & Oringer, Kenneth, "Synthesis of pure 3,3'-diaminobenzidine", issued 1976-03-09, assigned to Celanese Corporation