The formazans are compounds of the general formula
-N=N-C(R')=N-NH-R" formally derivatives of formazan
2NN=CHN=NH">2NN=CHN=NH unknown in free form.
Formazan dyes are artificial
chromogenic
In chemistry, the term chromogen refers to a colourless (or faintly coloured) chemical compound that can be converted by chemical reaction into a compound which can be described as "coloured". There is no universally agreed definition of the term. ...
products obtained by reduction of tetrazolium salts by
dehydrogenase
A dehydrogenase is an enzyme belonging to the group of oxidoreductases that oxidizes a substrate by reducing an electron acceptor, usually NAD+/NADP+ or a flavin coenzyme such as FAD or FMN. Like all catalysts, they catalyze reverse as well as f ...
s 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 Bamberger is a Bavarian and Southern German toponymic surname, and it indicates someone from Bamberg in Bavaria. Notable people with the surname include:
* Ana Maria Bamberger (born 1966), Romanian physician and playwright
* Ármin Vámbéry (bo ...
and Wheelwright independently. Their deep colour and
redox
Redox (reduction–oxidation, , ) is a type of chemical reaction in which the oxidation states of substrate (chemistry), substrate change. Oxidation is the loss of Electron, electrons or an increase in the oxidation state, while reduction ...
chemistry derive from their
nitrogen
Nitrogen is the chemical element with the symbol N and atomic number 7. Nitrogen is a nonmetal and the lightest member of group 15 of the periodic table, often called the pnictogens. It is a common element in the universe, estimated at se ...
-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
Deprotonation (or dehydronation) is the removal (transfer) of a proton (or hydron, or hydrogen cation), (H+) from a Brønsted–Lowry acid in an acid–base reaction.Henry Jakubowski, Biochemistry Online Chapter 2A3, https://employees.csbsju.ed ...
, the formed anion (3) is stabilized by
resonance
Resonance describes the phenomenon of increased amplitude that occurs when the frequency of an applied periodic force (or a Fourier component of it) is equal or close to a natural frequency of the system on which it acts. When an oscillatin ...
. With
transition metal
In chemistry, a transition metal (or transition element) is a chemical element in the d-block of the periodic table (groups 3 to 12), though the elements of group 12 (and less often group 3) are sometimes excluded. They are the elements that can ...
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 ...
).
![Formazan](https://upload.wikimedia.org/wikipedia/commons/0/05/Formazan.Tautomerism.svg)
Due to their ability to react with both strong acids and bases, formazans can be considered
amphoteric
In chemistry, an amphoteric compound () is a molecule or ion that can react both as an acid and as a base. What exactly this can mean depends on which definitions of acids and bases are being used.
One type of amphoteric species are amphiproti ...
.
Oxidation
Redox (reduction–oxidation, , ) is a type of chemical reaction in which the oxidation states of substrate change. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is the gain of electrons or a d ...
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:
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 compounds with
aldehyde
In organic chemistry, an aldehyde () is an organic compound containing a functional group with the structure . The functional group itself (without the "R" side chain) can be referred to as an aldehyde but can also be classified as a formyl grou ...
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 aldehyde ...
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
Tetrazene is a chemical compound with the molecular formula H2NN=NNH2. It is a colorless explosive material. An analogue is the organosilicon derivative (tms)2NN=NN(tms)2 where tms is trimethylsilyl. Isomeric with tetrazine is ammonium azide.
T ...
, which then forms a 3-substituted formazan.
Formazans can also be produced by the
oxidation
Redox (reduction–oxidation, , ) is a type of chemical reaction in which the oxidation states of substrate change. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is the gain of electrons or a d ...
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
Ethyl formate is an ester formed when ethanol (an alcohol) reacts with formic acid (a carboxylic acid). Ethyl formate has the characteristic smell of rum and is also partially responsible for the flavor of raspberries. It occurs naturally in th ...
or
orthoformate reacts with two equivalents of
phenylhydrazine
Phenylhydrazine is the chemical compound with the formula . It is often abbreviated as . It is also found in edible mushrooms.
Properties
Phenylhydrazine forms monoclinic prisms that melt to an oil around room temperature which may turn yellow ...
to yield 1,5-diphenylformazan, under
acidic conditions. Under basic conditions,
ethyl nitrate
Ethyl nitrate is the ethyl ester of nitric acid and has the chemical formula . It is a colourless, volatile, explosive, and highly flammable liquid. It is used in organic synthesis and as an intermediate in the preparation of some drugs, dyes ...
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
Amyl nitrite is a chemical compound with the formula C5H11ONO. A variety of isomers are known, but they all feature an amyl group attached to the nitrite functional group. The alkyl group is unreactive and the chemical and biological properti ...
.
Additionally, formazans can be obtained by the
decomposition
Decomposition or rot is the process by which dead organic substances are broken down into simpler organic or inorganic matter such as carbon dioxide, water, simple sugars and mineral salts. The process is a part of the nutrient cycle and is e ...
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
![MTT test formazan](https://upload.wikimedia.org/wikipedia/commons/0/01/MTT_test_formazan.jpg)
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](https://upload.wikimedia.org/wikipedia/commons/1/1a/Tetrazolium_reduction.svg)
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
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 ...
.
# 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.
#
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 or through direct reaction with
NADH
Nicotinamide adenine dinucleotide (NAD) is a coenzyme central to metabolism. Found in all living cells, NAD is called a dinucleotide because it consists of two nucleotides joined through their phosphate groups. One nucleotide contains an aden ...
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
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