Formazan sugar

The editors note with regret the passing of Laszld Mester on February 23, 1986. Mester worked extensively on hydrazine derivatives of sugars, and he contributed a notable article on the formazan reaction in Volume 13 of this series. 

Acyclic sugar hydrazones react with benzenediazonium chloride to give red formazans. When oxidized with N-bromosuccinimide (or, after acetylation, with lead tetraacetate), these yield tetrazolium salts. 

Various experiments made in this institute in recent years ( S, 10-lS) showed that the formazan reaction lent itself readily to studies of the structure of oxidized sugar derivatives whenever free aldehyde groups were present. Obviously, it was a useful reaction for investigations into the structure of ozone-oxidized carbohydrates, among them the sugar alcohols that had been studied by Harries. 

The present work showed, in perfect agreement with Harries, that on ozonizing sugar alcohols in an aqueous solution, they begin to oxidize and aldoses arise in the course of the oxidation process. For instance, n-mannose was prepared from ozone-oxidized D-mannitol with the aid of the formazan reaction, n-mannose was then identified as D-manno-V,V -diphenyl formazan (8). 

Other sugar alcohols, such as n-sorbitol and n-dulcitol, give an aldose phenylhydra-zone which is hard to isolate and convert to the formazan. Therefore, the aldose (n-glucose from n-sorbitol) was transformed into glucosazone, which was identified with the aid of the formazan reaction as n-glucosazone formazan (4). 

From ozone-oxidized sugar alcohols no formazan derivatives have yet been isolated in which the primary alcoholic hydroxyls, presumably present at both ends of the molecule, have oxidized to aldehydes. However, bisformazans, conspicuous for their capacity to crystallize and their insolubility in water, have been successfully prepared by other means (13). 

Polysaccharides not containing a free primary alcoholic group—e.g., xylan— have failed to yield formazan upon oxidation with ozone. This finding and the observations made in the present work in connection with ozone-oxidized sugar alcohols seem to support the suggestion that the formazan group takes the place of the primary alcoholic group. 

Like the sugar formazans (9), the formazans prepared from ozone-oxidized polysaccharides readily oxidize with A-bromosuccinimide to the corresponding colorless tetrazolium compounds. 

As in the formazans of the simple sugars (15), those of the ozone-oxidized polysaccharides undergo reductive disintegration by the action of hydrogen sulfide. Thus, the formazan of ozone-oxidized starch gives the corresponding thionic acid phenyl-hydrazide. 

VIII. The Formazan Reaction of Periodate-oxidized Sugar Derivatives. 148... 

It seems surprising that, during the 60 years which had elapsed since the first compounds in this group were reported, and despite the work of many investigators having a variety of objectives in formazan chemistry, no attempts to prepare the formazans of sugars had been described. 

These sugar compounds show all the characteristic properties of the formazans a brilliant red color, complexing with salts of heavy metals, and ready oxidation to colorless compounds. Nevertheless, further evidence was sought, in order to prove that they really belong to the formazan group. According to all current views on the mechanism of formazan formation, secondary phenylhydrazones, such as a 2-methyl-2-phenyl-... 

As has been pointed out in Section I, 2, when R and R" differ, the two isomers (A and B) permitted by the classical formulation cannot be isolated (because of the chelated structure of the formazans). This phenomenon, so very characteristic of the formazans, is also encountered in the formazans of sugars. The mono-p-bromo derivative of n-galactose diphenylformazan was prepared by two routes one was by causing n-galactose p-bromo-phenylhydrazone to react with diazotized aniline the other, by coupling D-galactose phenylhydrazone with diazotized p-bromoaniline as follows. 

Accordingly, the identity of the two products affords decisive evidence for the formazan structure of the new sugar derivatives. "... 

Since they contain no active methine groups, - the fcetose phenyl-hydrazones do not yield formazans this lack of reaction is therefore suitable for distinguishing ketoses from aldoses. It is more specific than the osazone test, frequently used in the identification of sugars, for it yields a different formazan from each aldose, whereas the osazone test does not differentiate between epimers. 

For identifying aldoses, this reaction has been simplified. The sugar phenylhydrazone (derived from the reaction of the sugar with phenyl-hydrazine) is not isolated, but, after addition of pyridine, is immediately coupled to give the formazan. 

Experiments on the separation of mixtures of sugar formazans by means of chromatography are in progress. 

With the objective of obtaining a biological indicator less toxic than T.T.C., and in view of a new and gradually increasing interest in tetrazolium compounds, attempts have been made to prepare them by the oxidation of sugar formazans. ... 

Using oxidants generally employed for the oxidation of simple formazans (for example, amyl nitrite and lead tetraacetate), the sugar formazans... 

Next, the sugar portion sensitive to oxidation was protected by means of acetylation, and the penta-O-acetyl-n-galactose diphenylformazan was oxidized with lead tetraacetate. By the action of the calculated amount of the oxidant, penta-O-acetyl-n-galactose diphenyltetrazolium chloride CG.T.C. acetate) was obtained as characteristically shaped, colorless prisms upon addition of an appropriate quantity of hydrochloric acid. Owing to its ionic structure, this compound is quite soluble in water and is readily reduced to the corresponding acetylated formazan, which is red. Saponification of the penta-O-acetyl-D-galactose diphenyltetrazolium... 

Direct conversion of sugar formazans to the corresponding sugar tetra-zolium compounds has been made possible by using iV-bromosuccinimide, as recommended by Kuhn and Munzing. ... 

Reductive decomposition of sugar formazans with hydrogen sulfide leads to sugar derivatives containing nitrogen and sulfur. ... 

The experimental data allow the following interpretation of the mechanism. By the action of hydrogen sulfide, the sugar formazan (I) is reduced to a sugar hydrazidine derivative (II), which becomes protonized (III) in the course of a slow SnI process, the hydrazidine ion splits off phenylhydrazine, whereafter the arising carbonium ion (IV) unites in a rapid... 

The formazan reaction permits an interpretation of the mutarotation curves of the sugar phenylhydrazones. i ... 

Until the formazan reaction was applied to this field, the finer structure of the sugar phenylosazones, and, indeed, whether they are cyclic or acyclic, was uncertain. 

Coupled with diazo compounds in pyridine-alcohol solution, the sugar osazones fail to yield formazans. This may be due to one or both of the following reasons. 

In order to find the reason for the failure of formazan reaction, a model experiment was set up using 2-quinolinecarboxaldehyde phenylhydrazone whose structure, if the nitrogen of the quinoline be considered, is like that of a phenylosazone, with the six-membered chelate ring in it as proposed by Fieser and Fieser for sugar osazones. 

Analogous to the formazans obtained by loosening the chelate ring in the above manner, the sugar osazones of supposedly chelate structure were found to undergo reaction in alkaline alcohol and to give n-glucose phenylosazone formazan in dark-violet needles. Mild acetylation produces the black tetraacetate of this compound. 

The fact that the formazan is formed ehminates structures XVII, XVIII, and XXI, which do not meet the conditions for this reaction. This fimiishes dired evidence in place of the indirect evidence on which Hardegger and Schreier had discarded structures XVIII and XXI. The bicyclic structure XIX, and XX, proposed by Percival" can be dismissed on comparison of the ultraviolet spectra. This compound, after coupling, should contain the two chromophoric groups of the simple formazans, whereas osazone formazans contain three of them. Anhydro- D-glucose phenylosazone formazan and its diacetate display practically the same maxima (maxima at 335 and 405 ran and 340 and 405 m/x, respectively) as does the indisputably-acyclic pyruvaldehyde phenylosazone formazan (maxima at 335 and 410 m/t), and these peaks differ substantially from the values for the simple sugar for-... 

Since the use of the formazan reaction had confirmed both the open-chain structure and the presence of the earlier-postulated chelate ring for the sugar phenylosazones, it appeared necessary to revise the then-current interpretations of their mutarotation. ... 

The former product is the first sugar derivative to contain the formazan group at the highest-numbered carbon atom the latter belongs to a novel class of sugar formazans. 

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