The formazan reaction

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. 

Mester, L., The Formazan Reaction in Carbohydrate Research, XIII, 105-167... 

Reaction of the dialdehyde with phenylhydrazine gave a derivative which formed an amorphous diphenylformazan (which, further, gave a monoacetate). Structure (55) was proposed for the formazan. Reaction of the dialdehyde with (p-nitrophenyl) hydrazine gave an amorphous mono-hydrazone, which was hydrolyzed by acid to glyoxal bis[(p-nitrophenyl)-hydrazone) and glycerose (as pyruvaldehyde). Structure (56) was assigned... 

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). 

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

XI. The Formazan Reaction as a Means for Establishing the Structure of Polysaccharides. 162... 

The reaction mechanism of formazan formation has been studied by many researchers. The fundamental work of Busch and his coworkers and the studies by Hiinig and Boes, and by Hauptmann and Perissd, merit special mention. A host of other authors have endeavored to widen the sphere of application of the formazan reaction, and have extended it successfully to the 4,4-iV-diarylsemicarbazonesi and guanylhydrazones, -i respectively. 

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

It thus appears that the formazan reaction offers a method of tracing and interpreting optically-observable changes in structure. 

The formazan reaction, carried out at certain critical rotational values, also permits the interpretation of the structural changes observable during mutarotation of n-galacturonic acid phenylhydrazone (see Fig. 11). 

On the other hand, the amorphous D-mannose phenylhydrazone acetate described by Hofmann,and more recently by Stepanenko and coworker underwent reaction but failed to produce the readily crystalline D-mannose diphenylformazan pentaacetate obtained by the acetylation of D-mannose diphenylformazan. The amorphous, acetylated D-mannose phenylhydrazone has a wide melting-point range and appears to be a mixture of partially acetylated phenylhydrazones in which the presence of the aldehydo form can be readily demonstrated by the formazan reaction. 

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. 

It is known that the formazan reaction depends upon two conditions. The first is the presence of an aldehydo Schiff base (—CH=N—), a criterion which is fulfilled in aWe%do-phenylhydrazones, but not in fceto-phenylhydrazones or in phenylhydrazones of the cyclic hemiacetal forms of the aldoses. The second condition is the presence of free imino hydrogen in the phenylhydrazone group. If Fischer s open-chain structure is correct, the phenylhydrazone group at Cl satisfies both conditions, and the formazan reaction would be expected to proceed according to the following equation. 

In the model reaction carried out with this compound, it was found that, in pyridine solution, the chelate N —> H completely inhibited formazan reaction, which indicates that, in n-glucose phenylosazone, similar chelation may be responsible for the failure to form a formazan. On replacing the pyridine with alkaline ethanol (often used to catalyze the formazan reaction), the chelate ring was loosened and coupling occurred to give A, A -diphenyl-C-(2-quinolyl)formazan. 

The formazan reaction offers a means for clarifying the structure of the mixed A and mixed B osazones. The former was described by Voto6ek and Vondra6ek as acyclic n-glucose l-(iV -phenyl)-2-(Ar -methyl-A -phenyl)osazone, and the latter as acyclic n-glucose l-(A -methyl-i r -phenyl)-2-(V -phenyl)osazone. The Percivals assigned the same n-fruc-topyranose l-(Ar -phenyl)-2-(V -methyl-Ar -phenyl)osazone structure to the compounds, but differentiated them as probable syn and avli forms. 

The formazan reaction, again, was found suitable for clarifying these points. Diels anhydro- D-glucose phenylosazone, like n-glucose phenyl-osazone, does not react in pyridine solution, while in an alkaline ethanol medium an osazone formazan is obtained which crystallizes as black nee-dles. Mild acetylation of this compound yields the black diacetate of Diels anhydro- D-glucose phenylosazone formazan. Analysis showed both acetyl groups to be oxygen-linked. 

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. ... 

This is now confirmed by the formazan reaction. From ozone-oxidized D-mannitol, n-mannose can be isolated in the form of its iV,iV -diphenyl-formazan. Applying this reaction to ozone-oxidized polysaccharides (for example, cellulose or starch), their bright-red formazans are obtained. ... 

The formazan reaction has also proved suitable for demonstration of the presence of aldehyde groups in polysaccharides oxidized by other means, even where these groups are limited in number or are the products of some side reaction. 

Yackel and Kenyon and Maurer and Reiff were the first to oxidize cellulose with nitrogen dioxide to obtain a poly(glycosiduronic acid). In addition to the principal reaction, their investigations pointed to the possibility of a number of side reactions. The formazan reaction revealed the presence of aldehyde groups in cellulose wads transformed to... 

In combination with other methods, use of the formazan reaction facilitates the study of polysaccharides of unknown structure and determination of the linkages in them. For example, if, from water-soluble polysaccharides, water-insoluble, highly nitrogenous formazans are prepared after oxidation with periodic acid, the estimation of the results of the periodate oxidation is greatly facilitated, since the precipitate is free from interfering, non-trans-formed byproducts. Similar favorable features were observed by Barry and Hirst - on isolating phenylhydrazones and other nitrogenous con-... 

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