The formazans are compounds of the general formula -N=N-C(R')=N-NH-R" formally derivatives of formazan ₂NN=CHN=NH unknown in free form. Formazan dyes are artificial chromogenic products obtained by reduction of tetrazolium salts by dehydrogenases and reductases. They have a variety of colors from dark blue to deep red to orange, depending on the original tetrazolium salt used as the substrate for the reaction.

Structure and reactivity

Formazans are intensely colorful compounds characterized by the following structure: N=N-C(R)=N-NH- and are closely related to azo (−N=N−) dyes. Their structure was first defined in 1892, by von Pechmann and by Bamberger and Wheelwright independently. Their deep colour and redox chemistry derive from their nitrogen-rich backbone. Formazans have a high

tautomeric Tautomers () are structural isomers (constitutional isomers) of chemical compounds that readily interconvert. The chemical reaction interconverting the two is called tautomerization. This conversion commonly results from the relocation of a hyd ...

and conformational flexibility. Due to the two alternating double bonds in the backbone, formazans can exist in four possible isomeric forms: syn, s-cis (closed form); syn, s-trans (open form); anti, s-cis; and anti, s-trans (linear form). 1,5-disubstituted formazans can exist as two tautomers (1 and 2 in the image below). Upon deprotonation, the formed anion (3) is stabilized by resonance. With transition metal ions ( Cu²⁺, Co³⁺, Ni²⁺, Zn²⁺, etc), formazans form highly coloured complexes (

chelates Chelation is a type of bonding of ions and molecules to metal ions. It involves the formation or presence of two or more separate coordinate bonds between a polydentate (multiple bonded) ligand and a single central metal atom. These ligands are ...

Due to their ability to react with both strong acids and bases, formazans can be considered amphoteric. Oxidation of such compounds results in their conversion into colorless tetrazolium salts. Among the various oxidants used are mercuric oxide, nitric acid, isoamyl nitrite, N-bromo succinimide, potassium permanganate, lead tetra-acetate and t-butyl hypochlorite. Depending on the conditions, tetrazolium salts can be reduced to form tetrazolyl radicals or formazan: Formazans2Tetrazoles

Synthesis

There various synthetic methods for the

synthesis Synthesis or synthesize may refer to: Science Chemistry and biochemistry *Chemical synthesis, the execution of chemical reactions to form a more complex molecule from chemical precursors ** Organic synthesis, the chemical synthesis of organ ...

of formazans. The reaction of

diazonium compound Diazonium compounds or diazonium salts are a group of organic compounds sharing a common functional group where R can be any organic group, such as an alkyl or an aryl, and X is an inorganic or organic anion, such as a halide. General propert ...

s with aldehyde

hydrazone Hydrazones are a class of organic compounds with the structure . They are related to ketones and aldehydes by the replacement of the oxygen =O with the = functional group. They are formed usually by the action of hydrazine on ketones or aldehydes. ...

s is one of the most common procedures to produce formazans. Hydrazones, which are electron-rich compounds, react with diazonium salts either at a nitrogen or a carbon atom to produce formazans. Diazonium salts couple to the amine nitrogen in the hydrazone with displacement of a hydrogen to give the intermediate, which then rearranges to the formazan. Another form to synthesize formazans is by the reaction of active methylene compounds with diazonium salts. Diazonium salts add to active methylene compounds to form an intermediate azo compound, followed by the addition of a second diazonium salt (under more alkaline conditions), yielding tetrazene, which then forms a 3-substituted formazan. Formazans can also be produced by the oxidation of the corresponding hydrazidines, usually prepared via reaction of hydrazonyl halides with the appropriate

hydrazine Hydrazine is an inorganic compound with the chemical formula . It is a simple pnictogen hydride, and is a colourless flammable liquid with an ammonia-like odour. Hydrazine is highly toxic unless handled in solution as, for example, hydrazine ...

derivatives. For example, ethyl formate or

orthoformate In organic chemistry, an ortho ester is a functional group containing three alkoxy groups attached to one carbon atom, i.e. with the general formula . Orthoesters may be considered as products of exhaustive alkylation of unstable orthocarboxylic ...

reacts with two equivalents of phenylhydrazine to yield 1,5-diphenylformazan, under acidic conditions. Under basic conditions, ethyl nitrate reacts at the methylene position to yield 3-methyl-1,5-diphenylformazan, which can also be obtained from the reaction of phenylazoethane with isoamyl nitrite. Additionally, formazans can be obtained by the decomposition of substituted tetrazolium salts either photochemically or under the influence of

ascorbic acid Vitamin C (also known as ascorbic acid and ascorbate) is a water-soluble vitamin found in citrus and other fruits and vegetables, also sold as a dietary supplement and as a topical 'serum' ingredient to treat melasma (dark pigment spots) an ...

in an

alkaline In chemistry, an alkali (; from ar, القلوي, al-qaly, lit=ashes of the saltwort) is a base (chemistry), basic, ionic compound, ionic salt (chemistry), salt of an alkali metal or an alkaline earth metal. An alkali can also be defined as ...

medium.

Application

Tetrazolium salts and their formazan products are widely used in histochemical methods, especially in colorimetric viability assays. These procedures are based on the reduction of tetrazolium by mitochondrial dehydrogenase enzymes, which is carried inside living cells: Tetrazolium reduction

Leading examples of the most used tetrazolium salts include: # INT or 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride, which is water-insoluble. # MTT or 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide, which is water-insoluble and used in the MTT assay. # XTT or 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, which is water-soluble. # MTS or 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, which is water-soluble and used in the

MTS assay The MTT assay is a colorimetric assay for assessing cell metabolic activity. NAD(P)H-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present. These enzymes are capable of reducing the t ...

. # TTC or tetrazolium chloride or 2,3,5-triphenyl-2H-tetrazolium chloride, which is water-soluble. # NBT used in a diagnostic test, particularly for chronic granulomatous disease and other diseases of phagocyte function. When reduced in a cell, either

enzymatically Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different molecule ...

or through direct reaction with NADH or

NADPH Nicotinamide adenine dinucleotide phosphate, abbreviated NADP or, in older notation, TPN (triphosphopyridine nucleotide), is a cofactor used in anabolic reactions, such as the Calvin cycle and lipid and nucleic acid syntheses, which require NAD ...

, the classical tetrazolium salt, MTT, turns blue to purple and may form an insoluble precipitate. These formazan dyes are commonly used in cell proliferation and toxicity

assay An assay is an investigative (analytic) procedure in laboratory medicine, mining, pharmacology, environmental biology and molecular biology for qualitatively assessing or quantitatively measuring the presence, amount, or functional activity of a ...

s such as the EpiDerm and EpiSkin tests since they only stain living, metabolically active cells.

References

Dyes {{biochem-stub