3,3'-Diaminobenzidine

3,3′-Diaminobenzidine (DAB) is an organic compound with the formula (C₆H₃(NH₂)₂)₂. This derivative of benzidine is a precursor to polybenzimidazole, which forms fibers that are renowned for their chemical and thermal stability.^[1] As its water-soluble tetrahydrochloride, DAB has been used in immunohistochemical staining of nucleic acids and proteins.^[2]

3,3′-Diaminobenzidine

Names
Preferred IUPAC name [1,1′-Biphenyl]-3,3′,4,4′-tetramine
Other names [1,1′-Biphenyl]-3,3′,4,4′-tetraamine (not recommended) 3,3′,4,4′-Biphenyltetramine 3,3′,4,4′-Tetraamino-diphenyl
Identifiers
CAS Number	91-95-2 Y[chemspider] 7411-49-6 (4HCl) Y
3D model (JSmol)	Interactive image
Beilstein Reference	1212988
ChEBI	CHEBI:90994 N
ChEMBL	ChEMBL1396955
ChemSpider	6804 Y
ECHA InfoCard	100.001.919
EC Number	202-110-6
PubChem CID	7071
RTECS number	DV8750000 DV8753000 (4HCl)
UNII	2RV4T6KHQI Y 26I0SJP460 (4HCl) Y
UN number	2811
CompTox Dashboard (EPA)	DTXSID2036827
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)
Hazards
GHS labelling:
Pictograms
Signal word	Danger
Hazard statements	H341, H350
Precautionary statements	P201, P202, P281, P308+P313, P405, P501
NFPA 704 (fire diamond)	2 1 2
Lethal dose or concentration (LD, LC):
LD50 (median dose)	mouse, oral Acute: 1834 mg/kg.
Safety data sheet (SDS)	External MSDS
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa). N verify (what is YN ?) Infobox references

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.^[3]

Preparation

Diaminobenzidine, which is commercially available, is prepared by treating 3,3′-dichlorobenzidine with ammonia with a copper catalyst at high temperature and pressure, followed by acidic workup.^[4]

An alternate synthesis route involves the diacylation of benzidine with acetic anhydride under basic conditions:^[1]

(NH₂)C₆H₄C₆H₄(NH₂) + 2 (CH₃CO)₂O ⟶ (NHCOCH₃)C₆H₄C₆H₄(NHCOCH₃) + 2 CH₃COOH

The diacetylated compound then undergoes nitration with nitric acid to produce an ortho-dinitro compound due to the ortho-directing acetyl substituents:^[1]

(NHCOCH₃)C₆H₄C₆H₄(NHCOCH₃) + 2HNO₃ ⟶ (O₂N)(NHCOCH₃)C₆H₃C₆H₃(NHCOCH₃)(NO₂) + 2H₂O

The acetyl groups are then removed through saponification:^[1]

(O₂N)(NHCOCH₃)C₆H₃C₆H₃(NHCOCH₃)(NO₂) + 2NaOH ⟶ (O₂N)(NH₂)C₆H₃C₆H₃(NH₂)(NO₂) + 2NaOCOCH₃

The dinitrobenzidine compound is then reduced with hydrochloric acid and iron to produce 3,3′-diaminobenzidine:^[1]

(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.^[1]

Applications

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

Diaminobenzidine is oxidized by hydrogen peroxide in the presence of hemoglobin to give a dark-brown color. This color change is used to detect fingerprints in blood.^[5] The solubility of DAB in water allows for adaptability compared to other detection solutions which use toxic solvents.^[6] Improperly prepared tissue samples may give false positives.^[7] 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, 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.^[8] One other method uses complexes of injected biocytin with avidin or streptavidin, biotin, and then peroxidase.