Characterization of Osazones

Fischer s discovery of the phenylosazones of the sugars, a sequel to his discovery of phenylhydrazine itself, led him to his famous carbohydrate researches. He recognized that some of these osazones are difficult to characterize with exactness their melting points are really decomposition temperatures. The need for attainment of better precision in the identification of osazones has been a matter of concern in 

Isbell and Harriet L. Frush, J. Research Natl. Bur. Standards, 11, 649 (1933). 

These sugar osotriazoles show sharp melting points and rotations (with no mutarotation) and give promise of aiding the precise characterization of osazones. 

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The osotriazoles, which possess sharp melting points and show no muta-rotation, have been used for the characterization of osazones. Owing... 

The fourth student in this group, Stanford Moore, began his doctoral thesis research with Link in 1935. His work involved the development of a new, and still useful, procedure for oxidizing aldoses to the aldonic acids by potassium hypoiodite in methanol solution the resulting aldonic acids were then converted into nicely crystalline benzimidazole derivatives that proved more suitable for the characterization of carbohydrates than the classical osazones. This method, developed by Moore and Link (1940), was extended to the characterization of hexuronic acids, as their bis(benzimidazole)s, after oxidation to the glycaric acid (R. Lohmar, Dimler, Moore, and Link, 1942), of lactic acid (Moore, Dimler, and Link, 1941 Dimler and Link, 1942), and of ribose, fucose, and digitoxose (Dimler and Link, 1943). During these... 

Formation of Osazones.—Osazones are readily formed from 3-deoxy-o-erythro-hexosuloae in the presence or absence of acid. However, the only crystalline osazones that have been obtained are the (4-nitrophenyl)osa-zone, the (2,4-dinitrophenyl)osazone, and the (2,5-dichlorophenyl)-osazone. The first two are identical with the respective osazones prepared from 3-deoxy-D-n 5o-hexose. The last two derivatives yield crystalline triacetates.The high melting points of many of these derivatives are not satisfactory for characterization, but their high optical rotations are a useful criterion of purity. ... 

Deoxy-D-well studied as the o-erythro isomer. It has been prepared in two ways from the decomposition of l,l -(carboxymethylamino)bis(l-deoxy-D-tagatose), ( di-D-tagatosegly-cine ), and from iV-butyl-D-galactosylamine. " These two methods and the isolation procedures are similar to the corresponding ones described for the erythro isomer. The 3-deoxy-n-i/ireo-hexosulose (42) was obtained as a colorless, amorphous powder, [ ]d + 10° — + 12.7° (in water), and was characterized as osazones. The (2,5-dichlorophenyl)osazone and the (2,4-dinitrophenyl)osazone are crystalline, the latter yielding a triacetate. " ... 

When positive identifieation of a sugar is required, several criteria of identification must be applied. Chromatography employing two different solvent systems and the use of different spray reagents, which permit differentiation based on differences in color developed with particular sugars, can give important confirmatory evidence in identification. Finally, a specific colorimetric reaction or the isolation and characterization of the osazone or benzimidazole derivative should be used to substantiate the identification. 

The ultraviolet spectra of osazones have been intensively studied, and, as shown earlier (see p. 154), the characteristics favor assigning of an acyclic structure to osazones. The spectra of osazones are characterized by three maxima, at about X256, 308-314, and 395-399 m/jt, which aid in their recognition. ... 

Historically, the discovery of osazone formation marked a significant advance in the practical aspects of sugar chemistry. Sugars are well known for their reluctance to crystallize from syrups. Their osazones, however, readily form yellow aystals with sharp melting points, thus simplifying the isolation and characterization of many sugars, particularly if they have been formed as mixtures or are impure. 

With excess phenylhydrazine in aqueous media buffered with acetate buffer osazones are formed, which are more suitable for identification in spite of the fact that their melting or decomposition points depend on the rate of heating. On mild oxidation in acid media osazones are converted to triazoles, which are characterized by sharp melting points and thus serve for further characterization of the osazones. 

Hydrolysis of the diacetamides is effected by acids. Dilute hydrochloric, sulfuric and nitric acid have been used. An aldose with one carbon atom less than the original nitrile is then liberated from the combination. Only exceptionally can this aldose be isolated in pure condition without transforming it into an insoluble derivative in these instances, the method has been employed for preparative work. In most cases the sugar has been characterized as an osazone. The aldose, without great purification, has been employed successfully for reduction - or oxidation experiments. ... 

D-apiose (1), characterized by optical rotatory data, paper-chromatographic mobility, and the melting point of the (p-bromophenyl)-osazone and the diisopropylidene acetal.20... 

The sugar osazones usually are crystalline and are useful for characterization and identification of sugars. Fischer employed them in his work that established the configuration of the sugars. The kind of information that can be obtained is illustrated by the following example ... 

Aldehyde content. The most logical evaluation of molecular weight by chemical means would be the estimation of terminal aldehyde, either by an absolute reducing value or by derivatization (as osazone formation or characterization as a carbonyl derivative following oxidation). 

An improved synthesis of dehydroascorbic acid has been reported (42). The oxidation of ascorbic acid in absolute methanol with oxygen over activated charcoal catalyst is reported to aflFord 28 in 95% yield. Dehydroascorbic acid has been characterized in solution as the monomer, 28 (43), and as the dimer (44,45) and its tetra acetyl derivative 29 (46). Several studies of mono- and di-hydrazone (48-53) and osazone (54) derivatives of dehydroascorbic acid have been reported. Hydrazone derivatives of dehydroascorbic acid have been used in the reductive synthesis of 2,3-diaza-2,3-dideoxy- and 2-aza-2-deoxyascorbic acid derivatives 30, 31, and 32 (55,56). Recently the reaction product of dehydro-L-ascorbic acid and L-phenylalanine in aqueous solution has been isolated and identified as tris(2-deoxy-2-L-ascorbyl)amine, 33, based on spectral and chemical data and its symmetry properties (57). 

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