Formazan, triphenyl

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. 

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. 

The 1,5-diphenyl and 1,3,5-triphenyl derivatives were discovered almost simultaneously by H. von Pechmann [38] and E. Bamberger [39] in 1892. Interest in those compounds was renewed in 1939 when G. Lakon [40] and R. Kuhn and D. Jerchel [41] found that colorless tetrazolium salts were very sensitive to biological reduction processes and were converted to deeply colored formazans ... 

Anodic oxidation of formazanes in MeCN-TEAP at a platinum anode produces tetrazolium salts in good yield [120, 121]. The ring closure probably takes place through the dication. A similar anodic coupling is the oxidation of 2-acetylpyridine phenyl-hydrazone to 3-methyl-l-phenyl-8-azaindazolium perchlorate [41], of benzilmonoxime phenylhydrazone to 2,4,5-triphenyl-1,2,3-triazole-1-oxide, and of 3-hydroxyiminomethyl-eneamino-6-chloropyridazine to 6-chloro-5-triazole[l,5-Z>]pyridazine-3-oxide [74, 122]. 

It has long been known that oxidation of simple formazans gives tetrazolium compounds which, even under biological conditions, are easily reduced once more to red formazans. For instance, triphenyltetrazolium chloride, prepared by oxidizing triphenyl formazan, is an approved biological indicator. 

Use In germination and viability tests. Viable parts of seed are stained red by deposition of red insoluble triphenyl formazan. 

Solvated nearly colorless needles from alcohol or chloroform, dry at 105°. Turns yellow on exposure to light Dec 243. Sol in water, alcohol, acetone insol in ether. Oxidizes aldoses and ketoses, as well as other a-ketols, and is thereby reduced to a water-insoluble, deep red pigment, a triphenyl-formazan. LDjg i.v. in mice 5600 ug/kg, RTECS Vol. II, R, J. Lewis, R. L, Tatken, Eds. (1980) p 690. 

The zones or spots of separated compormds are generally visualized by detecting dehydrogenase activity with tetrazolium salt-based reagents. The metabolically active bacteria convert the tetrazolium salt into red formazan dye (2,3,5-triphenyl-2H-tetra-zolium chloride, tetrazolium red). A number of tetrezolium salts were evaluated as potential substrates for the enzymatic reaction, but p-iodonitrote-trazolium violet, tetranitro blue tetrazolium, and MTT were found to be suitable substrates. The equations below show the principle of the reaction with tetrazolium salt, 3-[4,5-dimethylthiazol-2-yl]-2,5 dip-henyltetrazolium bromide (MTT, tetrazolium dye). 

To enhance the applicability of UV spectrophotometry in pharmaceutical steroid analysis by increasing its selectivity and sensitivity, various methods have been developed based on chemical reactions leading to colored derivatives. Although their importance has decreased considerably, some of these methods are still in use in pharmacopoeias, mainly in the assay of formulations. For example, the A" -3-oxo and A " -3-oxo steroids can be determined as iso-nicotinoyl hydrazones (/Uax 380 and 410 nm 11500 and 17000, respectively). The dihydroxy-acetone side chain of corticosteroids at C17 reduces tetrazolium reagents to colored formazans (Amax 485 and 525 nm with triphenyl tetrazolium chloride and Tetrazolium Blue reagents, respectively e 16000 and 24000), thus creating the basis for a stability-indicating indirect colorimetric assay. Corticosteroids... 

Various tetrazolium compounds, introduced into enzyme research by Kuhn and Jerchel (1941) and Lakon (1942), are reduced to scarlet-red (2,3,5-triphenyl-2H-tetrazolium), purplish (neoetrazo-lium) or blue (ditetrazolium) formazans, insoluble in water and soluble in fats. Formazan generation is practically inhibited by superoxide dismutase as well as by catalase (Bromme et al. 1999). When superoxide radical generation via the xanthine-xanthine oxidase system was measured by chemiluminescence an addition of nitroblue tetrazolium immediately abolished lucigenin-enhanced chemiluminescence. Nitroblue tetrazolium did not inhibit xanthine oxidase. 

Campbell et al. (1956) determined the polarographic reduction potentials of triphenyl tetrazohum, triphenyl formazan and benylidene. Based on Campbell s... 

Aniline diazotized at 0° with aq. NaNO in aq. HGl, the resulting diazonium salt soln. added dropwise with stirring at 0 to malonic acid in dimethyl-formamide-pyridine, and the product isolated after 4 hrs. ->N,N, G-triphenyl-formazan. Y 44%. F. e. s. F. A. Neugebauer and B. Kiichler, A. 706, 104 (1967). 

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