View source for Formazan

{{Short description|Class of chemical compounds; intensely colorful dyes}}
The '''formazans''' are compounds of the general formula [R-N=N-C(R')=N-NH-R"], formally derivatives of '''formazan''' [H<sub>2</sub>NN=CHN=NH], unknown in free form.<ref>[https://goldbook.iupac.org/terms/view/F02486 formazans] {{Webarchive|url=https://web.archive.org/web/20220120172844/https://goldbook.iupac.org/terms/view/F02486 |date=2022-01-20 }}. 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. {{ISBN|0-9678550-9-8}}. {{doi|10.1351/goldbook}}.</ref>
Formazan dyes are artificial [[Chromogen|chromogenic]] products obtained by reduction of '''tetrazolium salts''' by [[dehydrogenase]]s and [[Reductase|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-],<ref>{{Cite journal|last1=Khattab|first1=Tawfik|last2=Haggag|first2=Karima M|date=2017|title=Synthesis and spectral properties of symmetrical and asymmetrical 3-cyano-1, 5-diarylformazan dyestuffs for dyeing polyester fabrics|url=https://ejchem.journals.ekb.eg/article_4060.html|journal=Egyptian Journal of Chemistry|volume=60|issue=Conference Issue (The 8th International Conference of The Textile Research Division (ICTRD 2017), National Research Centre, Cairo 12622, Egypt.)|pages=33–40|doi=10.21608/ejchem.2017.1479.1103|issn=0449-2285|doi-access=free|access-date=2022-02-24|archive-date=2020-10-18|archive-url=https://web.archive.org/web/20201018162140/https://ejchem.journals.ekb.eg/article_4060.html|url-status=live}}</ref> and are closely related to [[Azo group|azo]] (−N=N−) dyes. Their structure was first defined in 1892, by [[Hans von Pechmann|von Pechmann]] and by [[Eugen Bamberger|Bamberger]] and Wheelwright independently.<ref>{{Cite book| publisher = Elsevier Science| isbn = 978-0-08-056272-8| last1 = Wolfrom| first1 = M.L.| last2 = Tipson| first2 = R.S.| title = Advances in Carbohydrate Chemistry| series = ISSN| date = 1958}}</ref><ref>{{Cite journal| doi = 10.1021/cr50002a004| issn = 0009-2665| volume = 55| issue = 2| pages = 355–483| last = Nineham| first = A. W.| title = The Chemistry of Formazans and Tetrazolium Salts| journal = Chemical Reviews| date = 1955-04-01|doi-access=free}}</ref> Their deep colour and [[redox]] chemistry derive from their [[nitrogen]]-rich backbone.<ref name="gilroy">{{Cite journal|last1=Gilroy|first1=Joe B.|last2=Otten|first2=Edwin|date=2020|title=Formazanate coordination compounds: synthesis, reactivity, and applications|journal=Chem. Soc. Rev.|volume=49|issue=1|pages=85–113|doi=10.1039/C9CS00676A|pmid=31802081|doi-access=free}}</ref>
Formazans have a high [[Tautomer|tautomeric]] and [[Conformational isomerism|conformational]] flexibility.<ref name="gilroy" /> 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).<ref>{{Cite journal|last1=Chang|first1=Mu-Chieh|last2=Roewen|first2=Peter|last3=Travieso-Puente|first3=Raquel|last4=Lutz|first4=Martin|last5=Otten|first5=Edwin|date=2014-12-10|title=Formazanate Ligands as Structurally Versatile, Redox-Active Analogues of β-Diketiminates in Zinc Chemistry|url=https://dx.doi.org/10.1021/ic5025873|journal=Inorganic Chemistry|volume=54|issue=1|pages=379–388|doi=10.1021/ic5025873|pmid=25493709|issn=0020-1669|access-date=2022-02-24|archive-date=2022-02-24|archive-url=https://web.archive.org/web/20220224105338/https://pubs.acs.org/doi/10.1021/ic5025873|url-status=live}}</ref>
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 stabilization|resonance]]. With [[transition metal]] ions ([[Copper|Cu<sup>2+</sup>]], [[Cobalt|Co<sup>3+</sup>]], [[Nickel|Ni<sup>2+</sup>]], [[Zinc|Zn<sup>2+</sup>]], etc), formazans form highly coloured complexes ([[Chelation|chelates]]).
[[File:Formazan.Tautomerism.svg|center|741x741px]] Due to their ability to react with both strong acids and bases, formazans can be considered [[amphoteric]].<ref>{{Cite journal| doi = 10.1139/v94-235| issn = 0008-4042| volume = 72| issue = 8| pages = 1849–1856| last1 = Katritzky| first1 = Alan R.| last2 = Belyakov| first2 = Sergei A.| last3 = Durst| first3 = H. Dupont| last4 = Xu| first4 = Ruixin| last5 = Dalal| first5 = Naresh S.| title = Syntheses of 3-(substituted)-2,4,6-triphenylverdazyls| journal = Canadian Journal of Chemistry| date = 1994-08-01| doi-access = free}}</ref>
[[Redox|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.<ref name="Shawali" /> Depending on the conditions, tetrazolium salts can be reduced to form tetrazolyl radicals or formazan:
[[File:Formazans2Tetrazoles.svg|center|601x601px]]
==Synthesis==
There various synthetic methods for the [[Chemical synthesis|synthesis]] of formazans.<ref name="Shawali">{{Cite journal|last1=Shawali|first1=Ahmad S.|last2=Samy|first2=Nevien A.|date=2015|title=Functionalized formazans: A review on recent progress in their pharmacological activities|journal=Journal of Advanced Research|volume=6|issue=3|pages=241–254|doi=10.1016/j.jare.2014.07.001|pmid=26257923|pmc=4522548|issn=2090-1232|doi-access=free}}</ref><ref>{{Cite book|last1=Karabach|first1=Y.Y.|title=Ligands: Synthesis, Characterization and Role in Biotechnology|last2=Kopylovich|first2=Maximilian|date=2014|publisher=Nova Science Publishers|isbn=9781631171437|pages=249–274|chapter=Synthesis, applications and coordination chemistry of formazans}}</ref>
The reaction of [[diazonium compound]]s with [[aldehyde]] [[hydrazone]]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.<ref name="chem">{{Cite journal|last=Daniel|first=DS|title=The Chemistry of Tetrazolium Salts|journal=Topics in Applied Chemistry|year=2002|pages=207–296|doi=10.1007/0-306-46906-5_7|isbn=0-306-45459-9}}</ref>
Another form to synthesize formazans is by the reaction of active [[Methylene group|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 [[Redox|oxidation]] 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|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 [[Amyl nitrite|isoamyl nitrite]].
Additionally, formazans can be obtained by the [[Chemical decomposition|decomposition]] of substituted tetrazolium salts either photochemically or under the influence of [[Vitamin C|ascorbic acid]] in an [[Alkali|alkaline]] medium.<ref>{{Cite book| publisher = Thieme| isbn = 978-3-13-178151-2| last1 = Charette| first1 = A.B.| last2 = Aggarwal| first2 = V.K.| last3 = Aitken| first3 = R.A.| last4 = Cicchi| first4 = S.| last5 = Cordero| first5 = F.| title = Science of Synthesis: Houben-Weyl Methods of Molecular Transformations Vol. 22: Three Carbon-Heteroatom Bonds: Thio-, Seleno-, and Tellurocarboxylic Acids and Derivatives; Imidic Acids and Derivatives; Ortho Acid Derivatives| date = 2014}}</ref>
==Application==
[[File:MTT test formazan.jpg|thumb|MTT test: Formation of formazan crystals from MTT in mesenchymal stem cells]]
Tetrazolium salts and their formazan products are widely used in histochemical methods, especially in colorimetric [[Viability assay|viability assays]].<ref>{{cite journal |vauthors=Gavanji S, Bakhtari A, Famurewa AC, Othman EM |title=Cytotoxic Activity of Herbal Medicines as Assessed in Vitro: A Review |journal=Chemistry & Biodiversity |volume=20 |pages=3–27 |date=January 2023 |issue=2 |pmid=36595710 |doi=10.1002/cbdv.202201098|s2cid=255473013 |doi-access=free }}</ref> These procedures are based on the reduction of tetrazolium by mitochondrial dehydrogenase enzymes, which is carried inside living cells:
[[File:Tetrazolium reduction.svg|400px|center]]
Leading examples of the most used tetrazolium salts include:<ref>{{cite journal |author=Altman FP |title=Tetrazolium salts and formazans |journal=Prog. Histochem. Cytochem. |volume=9 |issue=3 |pages=1–56 |year=1976 |pmid=792958|doi=10.1016/S0079-6336(76)80015-0 }}</ref>
#[[INT (chemical)|INT]] or 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride, which is water-insoluble.
#[[MTT assay|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 assay|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]].
#[[Tetrazolium chloride|TTC or tetrazolium chloride]] or 2,3,5-triphenyl-2H-tetrazolium chloride, which is water-soluble.
#[[Nitro blue tetrazolium chloride|NBT]] used in a diagnostic test, particularly for chronic granulomatous disease and other diseases of phagocyte function.
When reduced in a cell, either [[Enzyme|enzymatically]] or through direct reaction with [[NADH]] or [[NADPH]], the classical tetrazolium salt, MTT, turns blue to purple and may form an insoluble precipitate.<ref name="StockertJC 2018">{{cite journal |doi = 10.1016/j.acthis.2018.02.005 |pmid = 29496266 |title = Tetrazolium salts and formazan products in Cell Biology: Viability assessment, fluorescence imaging, and labeling perspectives |journal = Acta Histochemica |volume = 120 |issue = 3 |pages = 159–167 |year = 2018 |last1 = Stockert |first1 = Juan C. |last2 = Horobin |first2 = Richard W. |last3 = Colombo |first3 = Lucas L. |last4 = Blázquez-Castro |first4 = Alfonso |url = https://eprints.gla.ac.uk/158899/1/158899.pdf |access-date = 2022-02-24 |archive-date = 2022-01-20 |archive-url = https://web.archive.org/web/20220120131329/http://eprints.gla.ac.uk/158899/1/158899.pdf |url-status = live }}</ref><ref name="Stockert JC 2012">{{cite journal |doi = 10.1016/j.acthis.2012.01.006 |pmid = 22341561 |title = MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets |journal = Acta Histochemica |volume = 114 |issue = 8 |pages = 785–796 |year = 2012 |last1 = Stockert |first1 = Juan C. |last2 = Blázquez-Castro |first2 = Alfonso |last3 = Cañete |first3 = Magdalena |last4 = Horobin |first4 = Richard W. |last5 = Villanueva |first5 = Ángeles }}</ref> These formazan dyes are commonly used in cell proliferation and toxicity [[assay]]s such as the EpiDerm<ref>{{citation|url=https://www.mattek.com|title=Mattek|access-date=2022-02-24|archive-date=2022-02-08|archive-url=https://web.archive.org/web/20220208121211/https://www.mattek.com/|url-status=live}}</ref> and EpiSkin tests since they only stain living, metabolically active cells.<ref>{{cite journal |vauthors=Marshall NJ, Goodwin CJ, Holt SJ |title=A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function |journal=Growth Regul. |volume=5 |issue=2 |pages=69–84 |date=June 1995 |pmid=7627094}}</ref><ref>{{cite journal |vauthors=Scudiero DA, Shoemaker RH, Paull KD |title=Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines |journal=Cancer Res. |volume=48 |issue=17 |pages=4827–33 |date=1 September 1988 |pmid=3409223 |url=https://cancerres.aacrjournals.org/cgi/pmidlookup?view=long&pmid=3409223 |display-authors=etal |access-date=24 February 2022 |archive-date=24 February 2022 |archive-url=https://web.archive.org/web/20220224105338/https://aacrjournals.org/cgi/pmidlookup |url-status=live }}</ref>
==References==
{{Reflist}}
[[Category:Dyes]]