Formazans tetrazolium salts

In order to illustrate the application of LSV in mechanistic analysis we can look at the redox behavior of the formazan-tetrazolium salt system which we studied some years ago [17], 1,3,5-Triphenyl formazane was oxidized at controlled potential in CH3CN-Et4NC104 solution to 2,3,5-triphenyl tetrazolium perchlorate which was then isolated in quantitative yield. Coulometry showed that the overall electrode reaction was a two-electron oxidation. It has been shown that the rate of variation of Ep with log v was 30 mV per decade of sweep rate and that there was no variation of the peak potential with the concentration of 1,3,5-triphenylformazan. According to Saveant s diagnostic criteria (Table 1), four mechanistic schemes were possible e-C-e-p-p, e-C-d-p-p, e-c-P-e-p and e-c-P-d-p. If cyclization is the rate-determining step, then the resulting e-C-e-p-p and e-C-d-p-p mechanisms would not imply variation of Ep with the concentration of base. However, we have observed the 35 mV shift of Ep cathodically in the presence of 4-cyanopyridine as a b e. These observations ruled out the first two mechanisms. The remaining possibilities were then e-c-P-e and e-c-P-d, as shown in Scheme 3. 

The diazonium salts precursors can be aniline, o- and p-toluidine, o-and p-anisidine, o- and p-phenetidine. or 3-naphthy]amine. The resulting formazans are crystalline and inlensel> colored. They are soluble in organic solvents, giving a red-violet coloration that darkens to blue. Dehydrogeneration gives the corresponding tetrazolium salts, which are isolated as perbromides (Scheme 51. Table X-13). 

Tetrazolium salts Formazan dyes are produced on reaction of [33]... 

Although the formation of tetrazolium salts of structure 92 by the oxidation of formazans under acid conditions has been known for many years, it is only recently that the quatemization of 1,5-disubstituted... 

NADPH-diaphorase activity is the ability of an enzyme to reduce soluble tetrazolium salts to an insoluble, visible formazan. This activity is being used by many laboratories to localize NO synthase histochemically. 

Tetrazolium salts are an important class of leuco dyes that have found a wide range of applications. They are unique among the synthetic leuco dyes in that the colored form of the dye is the reduced form rather than the oxidized form. There are at least three extensive reviews on tetrazolium salts and the corresponding formazan dyes1-3 and one textbook.96 This chapter will describe some recent developments as well as some of the material covered in these reviews. 

Tetrazolium salts to a large extent are prepared from the corresponding formazans and to a lesser extent from tetrazoles. Therefore, synthetic methods for both formazans and tetrazoles will be discussed. Also discussed will be some properties of formazan dyes that influence the choice of the tetrazolium salt for any particular application. 

Tetrazolium salts are derivatives of the H-tetrazole (1) or 2H-tetrazole (2). The CA numbering systems for tetrazoliums and formazans are shown in 3 and 4. It should be noted that the carbon atoms in 3 and 4 are... 

In general, tetrazolium salts are prepared by (a) oxidation of formazans and (b) alkylation of tetrazoles. Therefore, synthetic methods for formazans and tetrazoles are discussed first, followed by some direct and miscellaneous methods. 

A variety of oxidizing agents have been used to effect the transformation of formazans to tetrazolium salts. The choice of the reagent is still empirical and depends on the solubility properties of the formazan as well... 

The earliest oxidations were effected with nitrous fumes and later with mercuric oxide and isoamyl nitrite.74 Lead tetraacetate in acetic acid is in many cases the reagent of choice, but the removal of by-products can present some difficulties.75 IV-Haloimides and amides in alcoholic solutions have been reported to yield essentially pure tetrazolium salts76 and have been found specially useful in the preparation of heteroaryl-substituted tetrazolium salt.77,78 The novel formazans 49 have been successfully oxidized to 50 using 7V-chloro succinimide (Eq. II).79 tert-Butyl hypo-... 

Electrochemical oxidation has also been reported to result in good yields of tetrazolium salts.97,98 Treatment of formazans with boron trifluor-... 

Some of the methods used for the preparation of either formazans or tetrazoles can lead directly to tetrazolium salts when appropriate substituents are present. 

Ferricyanide oxidation of 1,5-disubstituted thiocarbazones (116) give the mesoionic tetrazolium salts (117) under mild conditions (Eq. 21).190 This is in contrast to the strongly alkaline oxidation of carbazides leading to mercapto formazans as shown in Eq. 8 (Section 7.3.1.4). The heterocyclic triazine (118), obtained by the action of a diazonium salt on 2-... 

Tetrazolium salts are unstable in basic solutions yielding intense colors. This reaction is still little understood.233,234 In the reaction of 2,3,5-triphenyltetrazolium with hydroxide, it is postulated that a hydroxide ion is involved first as a counterion later leading to the hypothetical A-hydroxy-formazan (147).229 Weiner studied the kinetics of this reaction and identified 1,3,5-triphenylformazan in 10% yield. In concentrated alkaline solutions, the A-hydroxytetrazole (148) has been isolated from triphenyltetrazolium chloride (Scheme 19).235,236... 

Solutions of tetrazolium salts, e.g., 53, have been reported to both become colored and bleached under the influence of both UV and visible light. Several workers have attributed this phenomenon to photoreduction to the corresponding formazan (51) and the formation of a fluorescent colorless compound (152) through photooxidation.240- 243 The reduction of 152 under UV or blue light to the intense green radical structure (153) has also been reported (Scheme 21).244 A one-electron reduction product (154) is proposed as a short-lived intermediate in the photoreduction.245... 

In a related manner, gamma radiolysis of tetrazolium salts also yields formazans.256,257 Pulse radiolysis in aqueous solutions leads to tetrazole... 

This is by far the most important reaction of tetrazolium salts and accounts for the bulk of their many applications. A large variety of reagents can reduce tetrazolium salts, e.g., 53 to formazans, e.g., 51. Ascorbic acid, hydrazine, and hydroxylamine have been recommended for the preparation of formazans from tetrazolium salts.245 Stronger reducing agents such as ammonium sulfide, sodium amalgam, sodium dithionite, and catalytic hydrogenation can further reduce the formazans to the amidrazones, e.g.,... 

Electrochemical methods have been used extensively to elucidate the mechanism of reduction of tetrazolium salts. In aprotic media, the first step is a reversible one-electron reduction to the radical 154 as confirmed by ESR spectroscopy.256,266 As shown in Scheme 26, this radical can then disproportionate to the tetrazolium salt and the formazan anion (166) or take up another electron to the formazan dianion (167). The formation of the dianion through a direct reduction or through the intermediate tetrazolyl anion (168) has also been proposed.272-28 1,294 In aqueous solutions, where protonation/deprotonation equilibria contribute to the complexity of the reduction process, the reduction potentials are pH dependent and a one-electron wave is seldom observed. 

The physical and chemical properties of formazans have been reviewed in detail.1,2 In this discussion only those properties that directly or indirectly affect the choice of a tetrazolium salt as a leuco dye will be discussed. Spectral, acid—baseyedox, and metal complexing properties will be stressed. 

As discussed in Section 7.4.2.5, the reduction of tetrazolium salts to formazans often results in further reduction products. As seen in Scheme 24, reduction of formazans with ammonium sulfide leads to the hydrazidine 161. The reduction can proceed further eliminating an arylamine, yielding an amidrazone, e.g., 162.364 By contrast, alcoholic hydrogen sulfide attacks... 

A few electrochemical reductions of formazans to hydrazidine have been reported.370,372 Ho wever, as discussed in Section 7.4.2.6, electrochemical techniques have been widely used to study the redox chemistry of tetrazolium salts and formazans. Opinions about the reversibility of the electron transfer step, the number of electrons involved, and the identity of the rate-determining step differ widely.369- 371,656 The electrochemical oxidation of some novel formazans, e.g., 215 produces the dicationic species 216 (Eq. 28). The mechanism is not clearly understood.372,373... 

Triphenylformazan behaves as a bidentate ligand forming 2 1 complexes (217) with divalent copper, nickel, and cobalt.377 Formazan metal complexes can be compared to complexes of azo dyes or beta diketones due to structural similarity.301,302 In general, formazan metal complexes have low stability toward acids. However, when electron-donating substituents are added to the aromatic ring, a considerable enhancement in stability is observed. Cationic complexes of type 218 are also known. The complexation of formazan with metal cation can be accompanied by oxidation to the tetrazolium salt and the formation of a complex... 

With few exceptions, by far most applications of tetrazolium salts (e.g., analytical, photographic, and biochemical) utilize their reducibility to the corresponding formazan dyes. Tetrazolium salts, due to their resistance to acid and oxidation and the presence of a positive charge, find use in other applications such as antistatic agents and phase transfer catalysts. Over the past two decades, there have been thousands of citations regarding the applications of tetrazolium salts. Most of these citations are patents with similar or overlapping and even identical claims. Any attempt at completeness would be futile. The applications are sorted where discernible, and the earliest or the broadest claims are cited. 

Most analytical uses of tetrazolium salts utilize their reducibility. The ability of the resulting formazan to coordinate to metal ions, causing a characteristic shift in the spectrum, is often utilized to determine the metal ions or to enhance the sensitivity of the detection. 

The ability of metal ions to form complexes with formazans is utilized to determine these ions either directly (for low valent reducing ions) or indirectly in the presence of a reducing agent. Among others, molybdenum(VI) and vanadium(V) have been determined using this method.442,443 Indirect methods have been reported for the analyses of substances that do not reduce tetrazolium salts. Examples include arsenic in nickel ores436 and traces of selenium.437 A method for the extraction and analysis of a number of metal ternary ion association complexes has been described.444 - 448... 

Formazans and their metal complexes are used as textile dyes by direct application. The in situ reduction of tetrazolium salts has not been used to introduce the dyes to their substrates. Treatment with triaryl mono and bis tetrazolium salts followed by a reducing agent such as ascorbic acid or thioglycolic acid has been claimed as a method of introducing formazans as permanent hair dyes.641 There are some references to their use as therapeutic agents.642 544... 

It is remarkable that more than a century after the discovery of tetrazoliums, they continue to have wide applications. In particular, the reductive ring opening reaction leading to formazans continues to receive a lot of attention. In 1993, there were 316 citations to tetrazolium salts in Chemical Abstracts, 23 of which were to their reduction. Yet, the same reaction that makes them a unique class of reduction indicator leuco dyes, may be a part of a broader phenomenon. 

A colorimetric version of the method uses a tetrazolium salt. The oxidation of the NADH is coupled to the reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide by a diaphorase (EC 1.6.4.3) and a deep blue formazan develops. The absorbance is read at a wavelength between 540 and 600 nm. This is more sensitive than the ultraviolet method but is more prone to interference, especially from any reducing agents present in the sample, which will result in a positive error. 

The disproportionation reaction (Eq. 2) of two tetrazolinyl radicals was studied by Umemoto [18a] and it was concluded that this reaction is a slow process, and therefore this process should also be ruled out as a fast d-step. Importantly, the difference between the redox potentials of cR and R (AEp = 0.25 V) favors the backward reaction. The feasibility of the backward reaction is substantiated by ESR experiments by Maender and Russell [20] who found that the mixture of formazan and tetrazolium salt gave rise to tetrazolinyl radicals. Finally, the solution electron transfer (Eq. 3) is possible as a homogeneous electron transfer (d-step) since it would be reasonable to expect that the redox potentials of the reacting species are very close. However, this reaction would imply dEp/dlogv slope of 19.7 mV (Table 1) which was not observed. Taking all the arguments into account it can be concluded that the mechanism shown in... 

Scheme 3. is the most likely reaction pathway for the anodic cyclization of formazan to tetrazolium salt. 

The chemical oxidation of 1,3,5-triarylformazans to tetrazolium salts was first accomplished in 1894 [127], Almost no attention was given to these compounds for about 50 years after their discovery. This situation began to alter markedly because of the application of tetrazolium salts in histochemical, pharmacological, and other biomedical research areas [128]. Specifically, the tetrazolium salt is reduced to a colored formazan derivative by reducing enzymes found only in metabolically active cells. Anodic transformation of for-mazans to tetrazolium salts was performed in acetonitrile solution using cotrolled potential electrolysis [17,129], In our view this reaction could be considered as a method of choice for the preparation of tetrazolium salts. The products were obtained in high yield and the electrolysis can be performed in a divided cell under constant current and decoloration of the solution indicates the end point of the reaction. Recently the anodic oxidation of formazans to tetrazolium salts was performed successfully in aqueous ethanol solution [130]. 

XTT assay is suitable for measuring cell proliferation, cell viability or cytotoxicity. The tetrazolium salts are converted into a coloured formazan product by cellular enzymes present in the mitochondria of a metabolically active cell. These enzymes are rapidly inactivated when a cell dies, and hence the activity of these enzymes can be used to monitor the viability of a cell. 

Modified tetrazolium salts such as XTT have recently become available. Viable cells convert these modified compounds to a water-soluble formazan. As a result, the solubilization step can be eliminated from the procedure. 

Phytochemical reduction of tetrazolium salts has been observed by Kuhn and Jerschel. If 2,3,5-triphenyl tetrazolium chloride is added to fermenting yeast the corresponding formazan is formed. Like most formazyl compounds it has a red color. 

Trisubstituted tetrazolium salts (57) are the oxidation products of formazans. They have been used to produce tetrazolinyl radicals by controlled reduction <73AG(E)455>. 7V-Alkyltetrazolium... 

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