Formazan reduction

Formazans react with salts of heavy metals to give stable complexes 233 and they are also oxidized by Ar-bromosuccinimide or (after acetylation) by lead tetraacetate to yield tetrazolium salts234-236 Reduction of the latter give back the formazans. Reductive decomposition of sugar formazans (79) with hydrogen sulfide leads to aldothionic acid phenylhydrazides (82), 237-240 Treatment of these with benzaldehyde in the presence of hydrochloric acid yields sugar thiadiazolines (83) (Scheme 19),237... 

Fish test sample working solutions are adjusted to 2 g TE/mL in RPMI medium and then serially diluted (8x) in RPMI medium in separate vials. 10 pL from each dilution is added in triplicate to the 24 +OA and 24 -OA wells. Similarly, CTX-1 standard (0.5 ng/mL) is serially diluted (8x) and 10 pL from each dilution added in triplicate to 24 +OA and 24 -OA wells of the toxin standard plate. The 96-well plate cultures are then incubated 22-24 h. The treatment medium is removed at 22-24 h by hand-flick, replaced with (3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyltetrazolium) (MTT) solution (0.83 mg/mL RPMI medium 60 pL/well), and incubated for 30 min. The MTT solution is removed from the plates after 30 min by hand-flick and replaced with DMSO (100 pL/well). The plates are agitated gently side-to-side to disperse the formazan reduction product of MTT before measuring absorbance at 570 nm (reference wavelength of 630 nm) using a microplate reader. 

The well-known triphenyltetrazolium chloride (TTC) reaction for the detection of a-ketolsteroids, pyridinium carbinols and pyridinium glycols can also be included here [20-23]. The chromophore system of the red-colored formazan dye produced by reduction of the TTC is composed of highly conjugated double bonds resulting from the combination of a phenylhydrazone group with an azo group ... 

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... 

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 chemistry of tetrazolium thiolates and other mesoionic tetra-zoliums has been extensively reviewed.297,298 Tho ugh there are no reports of the chemical reduction of thiolates to formazan-like structures, the polarographic reduction of the complex betaines (146) to formazans has been reported.655... 

Formazans are stable in alkaline solution. Alkaline hydrolysis of functionalities on formazans such as nitriles, esters, and amides leads to the acids (Section 7.3.1.1). The case of 3-nitroformazans (198) is unique. Reaction with hydroxide ion gives 3-hydroxy formazan (199) which can be readily oxidized to the tetrazolium betaine. In the presence of hydrosulfide, a reduction of the nitro group takes place giving 3-aminoformazan (200) with traces of the 3-mercaptoformazan (201), which by contrast is the main product when ammonium polysulfide is used (Scheme 30).45,346... 

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... 

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. 

Liu Y, Schubert D. Cytotoxic amyloid peptides inhibit cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MMT) reduction by enhancing MTT formazan exocytosis. J Neurochem 1997 69 2285-2293. 

Abe K, Saito H. Amyloid beta protein inhibits cellular MTT reduction not by suppression of mitochondrial succinate dehydrogenase but by acceleration of MTT formazan exocytosis in cultured rat cortical astrocytes. Neurosci Res 1998 31 295-305. 

Nitroblue tetrazolium (NBT, 3,3 -(3,3,-dimethoxy-l,l,-biphenyl-4,4 -diyl)bis-2-(4-nitrophe-nyl)-5-phenyl-2H-tetrazolium dichloride) is reduced by superoxide to formazan as a final product, which can be measured spectrophotometrically [73]. Although the rate constant for NBT reduction by superoxide is moderately high 5.88+0.12x 104 1 mol 1 s 1 [74], the formation of formazan is not a simple one-electron transfer process, and the final product is formed as a result of disproportionation of intermediate free radicals. Similar to cytochrome c, NBT is easily reduced by the other reductants that confines its application for superoxide detection. Moreover, similar to epinephrine, NBT free radical is apparently... 

Table 26. Cu and AOX contents before and after reduction of Formazan Blue wastewater...

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. 

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... 

The thioaldonic phenylhydrazides obtained by reduction of saccharide formazans condense with benzaldehyde to give 1,3,4-thiadi-azolines, characterized by their high optical rotatory power.234,235... 

Growth conditions in deep-well microtiter plates were optimized with respect to optimal expression of active enzymes (Fig. 2.2.1.1). The best results were obtained with an expression time of 20 h at 37 °C (Fig. 2.2.1.1, lanes 7-9). Subsequently, E. coli cells were enzymatically disrupted by lysozyme treatment, and the carboligase activity was monitored by a modified tetrazolium salt color assay [16], This color assay is based on the reduction of the 2,3,5-triphenyltetrazolium chloride (TTC) 13 to the corresponding formazan 15, which has an intense red color (Fig. 2.2.1.2A). Before screening ofa BFD variant library, substrates and products were tested in the color assay. Neither substrate, benzaldehyde 4 nor dimethoxy-acetaldehyde 8, reduced TTC 13 however, the product 2-hydroxy-3,3-dimethoxy-propiophenone 10 already caused color formation at low concentrations of 2.5-10 mM (Fig. 2.2.1.2B). Benzoin 12 as the product also gave a color change at a similar concentration (data not shown). 

The evidence that O is a product of the burst has already been summarized. Another direct consequence of the formation of O is the reduction of nitroblue tetrazolium to form an insoluble deposit of blue formazan. The reduction of nitro blue tetrazolium had been observed to occur in PMNs in patients with bacterial infections. In intact cells this appears to be a consequence of the formation of O it fails to occur in PMNs from patients with chronic granulomatous disease which cannot undergo the respiratory burst it is diminished by anaerobiosis and it is inhibitable by superoxide dismutase... 

The electrochemistry of tetrazolium salts (220) in aqueous media was discussed in Part I. In a strictly aprotic medium 220 gives a reversible one-electron reduction to a radical with a lifetime longer than the time scale of CV.352 In moist acetonitrile the reaction becomes a two-electron reduction similar to that in aqueous, basic media a formazan (221) is formed... 

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