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
Skeletal formula of 3,3′-diaminobenzidine
Ball-and-stick model of the 3,3′-diaminobenzidine molecule
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
3D model (JSmol)
1212988
ChEBI
ChEMBL
ChemSpider
ECHA InfoCard 100.001.919
EC Number
  • 202-110-6
RTECS number
  • DV8750000
    DV8753000 (4HCl)
UNII
UN number 2811
  • 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 checkY
    Key: HSTOKWSFWGCZMH-UHFFFAOYSA-N checkY
  • 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
  • c2(c1cc(N)c(N)cc1)ccc(N)c(N)c2
Properties
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:
GHS08: Health hazard
Danger
H341, H350
P201, P202, P281, P308+P313, P405, P501
NFPA 704 (fire diamond)
NFPA 704 four-colored diamond
2
1
2
Lethal dose or concentration (LD, LC):
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 checkY☒N ?)
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 CH3CO2H

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) + 2(NaOCOCH3)

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

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.[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.

References
  1. Hans Schwenecke, Dieter Mayer "Benzidine and Benzidine Derivatives" in Ullmann's Encyclopedia of Industrial Chemistry, 2005, Wiley-VCH, Weinheim.
  2. Roschzttardtz, H.; Grillet, L.; Isaure, M.-P.; Conejero, G.; Ortega, R.; Curie, C.; Mari, S. (2011), "The Plant Cell Nucleolus as a Hot Spot for Iron", The Journal of Biological Chemistry, 286 (32): 27863–27866, doi:10.1074/jbc.C111.269720, PMC 3151030, PMID 21719700
  3. Hui-Fen Qian & Wei Huang (2010). "Biphenyl-3,3′,4,4′-tetraamine". Acta Crystallographica. E66 (5): o1060. doi:10.1107/S1600536810012511. PMC 2979072. PMID 21579117.
  4. US 3943175, Druin, Melvin L. & Oringer, Kenneth, "Synthesis of pure 3,3′-diaminobenzidine", issued 1976-03-09, assigned to Celanese Corporation
  5. "D.A.B. (Diaminobenzidine)". Chesapeake Bay Division, International Association for Identification. Archived from the original on 23 December 2007. Retrieved 2007-11-09.
  6. Sahs P (1992), "DAB: An Advancement in Blood Print Detection", J. Forensic Ident., 42: 412–420
  7. "Liquid DAB Substrate" (pdf). Dako. Retrieved 2008-02-02.
  8. Falangola, M. F.; Lee, S. P.; Nixon, R. A.; Duff, K. & Helpern, J.K. (2005). "Histological co-localization of iron in Abeta plaques of PS/APP transgenic mice". Neurochemical Research. 30 (2): 201–205. doi:10.1007/s11064-004-2442-x. PMC 3959869. PMID 15895823.
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