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The formazans are compounds of the general formula R-N=N-C(R')=N-NH-R", formally derivatives of formazan H2NN=CHN=NH, unknown in free form. formazans . In: Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997). Online version (2019-) created by S. J. Chalk. . .
Formazan dyes are artificial Chromogen products obtained by reduction of tetrazolium salts by and Reductase. 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.
Formazans have a high Tautomer 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 (Copper, Cobalt, Nickel, Zinc, etc), formazans form highly coloured complexes (Chelation).
Due to their ability to react with both strong acids and bases, formazans can be considered amphoteric.
Redox 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:
The reaction of diazonium compounds with aldehyde 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 group 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 Redox of the corresponding hydrazidines, usually prepared via reaction of hydrazonyl halides with the appropriate hydrazine derivatives. For example, ethyl formate or orthoformate reacts with two equivalents of phenylhydrazine to yield 1,5-diphenylformazan, under Acid. 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 Amyl nitrite.
Additionally, formazans can be obtained by the decomposition of substituted tetrazolium salts either photochemically or under the influence of Vitamin C in an Alkali medium. (2026). 9783131781512, Thieme. ISBN 9783131781512
When reduced in a cell, either Enzyme or through direct reaction with NADH or NADPH, 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 such as the EpiDerm and EpiSkin tests since they only stain living, metabolically active cells.
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